陈红霞,邵建柱,孙建设,曹晓凤,乔雪华

• 1:河北农业大学园艺学院

摘要(Abstract)：

【目的】为建立检测3种苹果潜隐性病毒更为快速准确的方法,【方法】以携带苹果茎沟病毒(ASGV)、苹果褪绿叶斑病毒(ACLSV)和苹果茎痘病毒(ASPV)的成龄苹果树韧皮部为试材,根据基因库中苹果茎痘病毒(Apple stempitting virus,ASPV)的外壳蛋白基因序列,设计合成了2对特异性引物,分别与合成的苹果茎沟病毒(ASGV)引物和苹果褪绿叶斑病毒(ACLSV)的引物组合配伍,筛选出最佳的引物对组合。对cDNA的合成所用引物和总RNA模板进行遴选和量化,对PCR过程中cDNA的用量、引物对的终浓度、退火温度等主要影响因素进行优化。【结果】结果表明,反转录引物为Oligo(dT)18、总RNA 0.1～3.0μL时,可以得到较好的cDNA;ASPV-FR与ASGV-Pch、ACLSV-Pch引物组合优于ASPV-Pch,并且筛选出4组最佳的终浓度处理组合;cDNA用量为2.0~4.0μL、退火温度为50.0~52.9℃、循环数为35时,扩增效果较好。采用优化的多重RT-PCR体系对采自陕西和山东两省的样品进行检测,并用3种病毒的单重RT-PCR体系进行验证。【结论】结果呈现高度一致性,充分印证了该多重RT-PCR体系的准确性,适用于大量样品的快速检测。

关键词(KeyWords)： 苹果病毒;苹果茎沟病毒;苹果茎痘病毒;苹果褪绿叶斑病毒;两步多重RT-PCR

Abstract:

Keywords:

基金项目(Foundation): 国家苹果产业技术体系项目(CARS-28)

作者(Author): 陈红霞,邵建柱,孙建设,曹晓凤,乔雪华

DOI: 10.13925/j.cnki.gsxb.2012.04.008

参考文献(References)：

• [1]WANG Guo-ping.Atlas of virus diseases of fruit plants in China[M].Beijing:The Jindun Publishing House,2001:10-11.王国平.中国果树病毒病原色图谱[M].北京:金盾出版社,2001:10-11.
• [2]WANG Guo-ping.Prevent and cure apple virus diseases[M].Beijing:The Jindun Publishing House,1994:3.王国平.苹果病毒病防治[M].北京:金盾出版社,1994:3.
• [3]CHENG Yu-qin,HAN Zhen-hai,XU Xue-feng.The technology ofdeleting and detecting virus in apple[J].Chinese Agricultural Sci-ence Bulletin,2003,19(1):72-74,96.程玉琴,韩振海,徐雪峰.苹果病毒及其脱毒检测技术研究进展[J].中国农学通报,2003,19(1):72-74,96.
• [4]XU Rong-xue,MING Jun,MU Ding,LIU Chun,TANG Geng-guo,WANG Xiao-wu.Detection of three lily viruses by multiplex RT-PCR[J].Acta Horticulturae Sinica,2007,34(2):443-448.徐榕雪,明军,穆鼎,刘春,汤庚国,王晓武.百合三种病毒的多重RT-PCR检测[J].园艺学报,2007,34(2):443-448.
• [5]DING Fang,CAO Qing,WANG Guo-ping,YI Gan-jun,ZHONGYun.Studies on the simultaneous detection of Citrus HuanglongbingPathogen,Citrus exocortis virus,Citrus tristeza virus by multiplexRT-PCR[J].Acta Horticulturae Sinica,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.
• [6]YANG Hong-yi,ZHANG Zhi-hong,LI Li-li,ZHAO Min.Detec-tion o f straw berry viruses by multiplex RT-PCR[J].Acta Phytopatho-logica Sinica,2007,37(5):549-552.杨洪一,张志宏,李丽丽,赵敏.利用多重RT-PCR技术检测草莓病毒的研究[J].植物病理学报,2007,37(5):549-552.
• [7]LI Wen-hui,NIU Jian-xin.The technology of apple viruses[J].Northern Fruits,2006,4:3-5.李文慧,牛建新.苹果病毒的研究现状[J].北方果树,2006,4:3-5.
• [8]KLERKS M M,LEONE G,LINDNER J L,SCHOEN C D,VANDEN HEUVEL J F J M.Rapid and sensitive detection of Apple stempitting virus in apple trees through RNA amplification and probingwith fluorescent molecular beacons[J].Phytopathology,2001,91:1085-1091.
• [9]ZHANG Qiang,NIU Jian-xin,LI Xi-ping.A study on the one-stepRT-PCR for detection of Apple chlorotic leaf spot virus[J].Acta A-griculturae Boreali-occidentalis Sinica,2006,15(4):67-69.张强,牛建新,李西萍.苹果褪绿叶斑病毒一步法RT-PCR检测[J].西北农业学报,2006,15(4):67-69.
• [10]MARINHO V L A,KUMMERT J,RUFFLARD G,COLINET D,LEPOIVRE P.Detection of Apple stem grooving virus in dormantapple trees with crude extracts as templates for one-step RT-PCR[J].Plant Disease,1998,82:785-790.
• [11]WANG Zhuang-wei.Study on apple latent virus elimination and de-tection technique[D].Nanjing:Nanjing Agricultural University,2006.王壮伟.苹果潜隐性病毒的检测与脱除技术研究[D].南京:南京农业大学,2006.
• [12]LI Wen-hui,NIU Jian-xin.Detection of apple stem grooving virusby reverse transcription polymerase chain reaction(RT-PCR)[J].Acta Agriculturae Boreali-occidentalis Sinica,2006,15(4):105-107.李文慧,牛建新.苹果茎沟病毒RT-PCR检测技术[J].西北农业学报,2006,15(4):105-107.
• [13]KLERKS M M,LEONE G,LINDNER J L.Rapid and sensitive de-tection of Apple stem pitting virus in apple trees through RNA am-plification and probing with fluorescent molecular beacons[J].Phy-topathology,2001,91(11):1085-1091.
• [14]KUNDU J K.The occurrence of apple stem pitting virus and applestem grooving virus within field-grown apple cultivars evaluated byRT-PCR[J].Plant Protection Science,2003,39:88-92.
• [15]RWAHNIH M A,TURTURO C,MINAFRA A,SALDARELLI P,MYRTA A,PALLAS V,SAVINO V.Molecular variability of applechlorotic spot virus in different hosts and geographical regions[J].Journal of Plant Pathology,2004,86(2):117-122.
• [16]KUNDU J K,SVOBODA J,POLANK J.Detection of apple stemgrooving virus in different tissues of apple trees throughout the year[J].Plant Protection Science,2003,39:93-96.
• [17]FAN Xu-dong,DONG Ya-feng,ZHANG Zun-ping,LI Li-li,PEIGuang-qian.Multiplex RT-PCR assay for simultaneous detection ofthree latent apple viruses[J].Acta Horticulturae Sinica,2009,36(12):1821-1826.范旭东,董雅凤,张尊平,李丽丽,裴光前.3种苹果潜隐病毒多重RT-PCR检测体系的建立[J].园艺学报,2009,36(12):1821-1826.
• [18]MENZEL,JELKMANN,MAISS.Detection of four apple viruses bymultiplex RT-PCR assays with coamplification of plant mRNA asinternal control[J].Journal of Virological Methods,2002,99:81-92.