河南省烟草马铃薯Y病毒体外特性及血清学检测技术的研究
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摘要
用生物学方法研究了河南省PVY的体外特性，结果表明：河南省PVY的稀释限点为10-6～10-7，致死温度为70～75℃，体外存活期为4～6d。体外存活期与原报道相符，稀释限点与致死温度均比已有报道要高。对PVY、CMV和TMV三者之间的相互作用进行了研究，结果表明，每两种病毒的混合侵染都会加重对寄主植物的危害，并且症状表现存在差别。
分别以三生烟和普通烟为繁殖材料，采用蔗糖垫超速离心法提纯病毒，结果表明病毒提纯物最大吸收峰在260nm处，最低吸收峰在250nm处，OD280/260分别为：三生烟0.840873，普通烟0.837676，提纯物显示出典型的病毒核蛋白体光谱吸收特征。
比较了ACP-ELISA和DAS-ELISA两种检测方法，结果前者比后者灵敏性好。对ACP-ELISA方法进一步的优化结果表明，样品最佳稀释倍数为5倍，最佳孵育时间为10h，一抗和二抗的最佳孵育时间分别为8h和4℃过夜，是比较理想的优化ACP-ELISA检测方法。
我们利用优化的ACP-ELISA检测技术，对PVY在河南省主要产烟区的分布及危害情况、PVY在病残体中的存活时间、病株上采收的种子、田间及田边的作物和杂草进行了研究。对在三大类症状表现所采病样分别用ACP-ELISA法检测其PVY携带情况。⑴表现为全株均有叶脉坏死症状，叶背面支脉变褐坏死，叶片出现疱斑，皱缩。检测PVY阳性率为100%。⑵症状表现为上部叶表现花叶症状，中下部
叶片有坏死、蚀刻纹症状。检测PVY阳性率为66.7%。⑶症状表现为混合型，上部叶表现花叶，下部叶表现橡叶纹。检测PVY阳性率为50%。说明PVY在河南省主要产烟区的发生非常普遍而严重。
PVY在病残体中的存活时间较长为110d，很有可能成为下一年的初侵染来源；经ACP-ELISA检测，烟田及田边的作物和杂草，如红薯、稗草、苋色藜、龙葵等均不带毒，病株上采收的种子带毒。通过这些研究将有利于我们有针对性的制定防治措施，为开展PVY的防治工作提供理论依据。
The characteristics in vitro of PVY isolated from tobacco in Henan Province were studied by biological methods. The results showed that (i)the Dilution End Point(DEP) of PVY in Henan Province was 10-6 – 10-7,(ii) its Thermal Inactivation Point (TIP)was 70癈 - 80癈,(iii) and its Longevity in vitro (LIV)was 4 -6 day,which is same to that reported before.But the DEP and TIP is higher .In the analysis of the interaction between PVY and CMV TMV, it was found that inoculation of virus-mixture can cause more serious symptoms than the single inoculation, and the symptom varies in different mixture.
PVY was purified from N.tobacum samsan and Nicotiana tobacum by using sucrose cushion centrifugation. The purified PVY has a peak of absorption at 260nm,and a lowest absorption at 250nm .The ratio of OD280/OD260 is 0.840873, 0.837676,from N.tobacum samsun and N. tobacum, respectively. The result shows typical absorbing characteristic of viral nucleoprotein.
Compared ACP-ELISA with DSA-ELISA, the result shows that the former is more sensitive than the later. We choose ACP-ELISA as the detection method and optimized its procedure, the sample's best dilution ratio is 5 times, the sample's best incubating time is lOh.the optimum incubating period for first antibody and second antibody are 8h and overnight at 4, respectively.
By using the optimized ACP-ELISA methord,we studied the distribution and the
damage of PVY in Henan Province , the livability of PVY in infected plant residue, the existence on the tobacco-seed gathered from the diseased plants and the neighboring crops and weeds in the tobacco field were infected by PVYThe result shows that PVY can damage tobacco plants seriously. Divided the disease sample into three types according to the symptoms and detected it by ACP-ELISA method ,The results demonstrated that (l)the veins of all infected plants show necrosis. The branch veins of leaf's reverse side become brown and necrotic; the leaf has dark green bleb and twist, the ratio of PVY positive reaction is 100%. (2)the top leaves are mosaic, the middle and bottom leaves show necrosis and etching veins, the ratio of PVY positive reaction is 66.7%(3)the top leaves are mosaic and the bottom leaves become oak leaf vein-like necrotic, the ratio of PVY positive reaction is 50%.The livability of PVY in diseased plant residue is llOd, therefore, the diseased plant residue is possibly the infectant source. The neighboring crops and weeds around the tobacco field do not carry PVYThe infected tobacco seeds are infected by PVY. Those research results are helpful for forecast and control of PVY on tobacco.
分别以三生烟和普通烟为繁殖材料，采用蔗糖垫超速离心法提纯病毒，结果表明病毒提纯物最大吸收峰在260nm处，最低吸收峰在250nm处，OD280/260分别为：三生烟0.840873，普通烟0.837676，提纯物显示出典型的病毒核蛋白体光谱吸收特征。
比较了ACP-ELISA和DAS-ELISA两种检测方法，结果前者比后者灵敏性好。对ACP-ELISA方法进一步的优化结果表明，样品最佳稀释倍数为5倍，最佳孵育时间为10h，一抗和二抗的最佳孵育时间分别为8h和4℃过夜，是比较理想的优化ACP-ELISA检测方法。
我们利用优化的ACP-ELISA检测技术，对PVY在河南省主要产烟区的分布及危害情况、PVY在病残体中的存活时间、病株上采收的种子、田间及田边的作物和杂草进行了研究。对在三大类症状表现所采病样分别用ACP-ELISA法检测其PVY携带情况。⑴表现为全株均有叶脉坏死症状，叶背面支脉变褐坏死，叶片出现疱斑，皱缩。检测PVY阳性率为100%。⑵症状表现为上部叶表现花叶症状，中下部
叶片有坏死、蚀刻纹症状。检测PVY阳性率为66.7%。⑶症状表现为混合型，上部叶表现花叶，下部叶表现橡叶纹。检测PVY阳性率为50%。说明PVY在河南省主要产烟区的发生非常普遍而严重。
PVY在病残体中的存活时间较长为110d，很有可能成为下一年的初侵染来源；经ACP-ELISA检测，烟田及田边的作物和杂草，如红薯、稗草、苋色藜、龙葵等均不带毒，病株上采收的种子带毒。通过这些研究将有利于我们有针对性的制定防治措施，为开展PVY的防治工作提供理论依据。
The characteristics in vitro of PVY isolated from tobacco in Henan Province were studied by biological methods. The results showed that (i)the Dilution End Point(DEP) of PVY in Henan Province was 10-6 – 10-7,(ii) its Thermal Inactivation Point (TIP)was 70癈 - 80癈,(iii) and its Longevity in vitro (LIV)was 4 -6 day,which is same to that reported before.But the DEP and TIP is higher .In the analysis of the interaction between PVY and CMV TMV, it was found that inoculation of virus-mixture can cause more serious symptoms than the single inoculation, and the symptom varies in different mixture.
PVY was purified from N.tobacum samsan and Nicotiana tobacum by using sucrose cushion centrifugation. The purified PVY has a peak of absorption at 260nm,and a lowest absorption at 250nm .The ratio of OD280/OD260 is 0.840873, 0.837676,from N.tobacum samsun and N. tobacum, respectively. The result shows typical absorbing characteristic of viral nucleoprotein.
Compared ACP-ELISA with DSA-ELISA, the result shows that the former is more sensitive than the later. We choose ACP-ELISA as the detection method and optimized its procedure, the sample's best dilution ratio is 5 times, the sample's best incubating time is lOh.the optimum incubating period for first antibody and second antibody are 8h and overnight at 4, respectively.
By using the optimized ACP-ELISA methord,we studied the distribution and the
damage of PVY in Henan Province , the livability of PVY in infected plant residue, the existence on the tobacco-seed gathered from the diseased plants and the neighboring crops and weeds in the tobacco field were infected by PVYThe result shows that PVY can damage tobacco plants seriously. Divided the disease sample into three types according to the symptoms and detected it by ACP-ELISA method ,The results demonstrated that (l)the veins of all infected plants show necrosis. The branch veins of leaf's reverse side become brown and necrotic; the leaf has dark green bleb and twist, the ratio of PVY positive reaction is 100%. (2)the top leaves are mosaic, the middle and bottom leaves show necrosis and etching veins, the ratio of PVY positive reaction is 66.7%(3)the top leaves are mosaic and the bottom leaves become oak leaf vein-like necrotic, the ratio of PVY positive reaction is 50%.The livability of PVY in diseased plant residue is llOd, therefore, the diseased plant residue is possibly the infectant source. The neighboring crops and weeds around the tobacco field do not carry PVYThe infected tobacco seeds are infected by PVY. Those research results are helpful for forecast and control of PVY on tobacco.
引文
[1]郑宪滨，谢德平，王廷晓等．某些因素对烤烟病毒病(PVY、CMV、TMV)发病程度的影响调查[J]．烟草科技，1997，(4)：40–42．
[2]郭兴启，朱汉城，吕士恩等．酶标记羊抗兔间接法检测烟草马铃薯Y病毒的研究[J]．中国烟草学报，1998，4(2)：38–42．
[3]周本国，高正良，刘小平．安徽省烟草脉斑病(PVY)的发生趋势及防治措施[J]．安徽农学通报，1998，4(1)：34–35．
[4]程宝玉，吴娟霞，陈卫华等．豫西烟草脉斑病发生规律及药剂防治研究[J]．烟草科技，2002，(7)：46–48．
[5]田波，裴美云．植物病毒研究方法[M]．北京：科学出版社，1987，42–43．
[6]王劲波，王凤龙，钱玉梅等．山东烟区主要病毒的株系鉴定[J]．中国烟草学报，1998，4(1)：24–30．
[7]谈文，蒋士君，刘骏等．烟草病理学教程[M]．北京：中国科学技术出版社，1995，129–131．
[8]董继超,李花婷,苏永士,等.河南省烟草主要病害流行规律分析[Ｊ].河南农业大学学报,1992,(26增刊):31-39.
[9]蒋金炜,蒋士君,董继超.烟草病虫害防治[Ｍ].郑州:河南科学技术出版社,1994.87-88.
[10]谈文.烟草病虫害防治手册[Ｍ].北京:金盾出版社,2000.116-118.
[11]马国胜,郭红,高正良,等.固镇县烟草脉斑病流行规律调查研究[Ｊ].烟草科技,2000,(2):46-47.
[12]李嵩震,马中仁,侯文华.河南烟草栽培[Ｍ].郑州:河南科学技术出版社,1997.61-65.
[14]彭宏梅，李月秋，张仲凯等.应用电镜技术研究大理州烟草苗期病毒病的发生状况，云南农业大学学报.2000，15（1）：70-71
[13]周艳玲，刘学敏，孟玉芹.马铃薯Y病毒的检测技术[J].中国马铃薯，2000.14(2)：89-93
[15]Ｅ.库尔斯塔克.植物病毒比较诊断指南,农业出版社,1991,518
[16]王秀芳，任广伟，王凤龙.烟草上马铃薯Y病毒的RT-PCR检测，中国烟草学报,2003，9(2)28-30
[17]吴红芝，孔宝华，陈海如等.昆明地区菊花病毒病的调查与鉴定. 云南农业大学学报，2002，17（1），24-27
[18]张鹤龄.酶联免疫吸附测定(ELISA)及其在植物病毒鉴定中的应用.马铃薯,1983,(4):35～41
[19]仲乃琴.ELISA技术检测马铃薯病毒的研究，甘肃农业大学学报，1998，33（2）：178-181
[20]李汝刚,郭军,鞠振林等.马铃薯Ｘ、Ｙ病毒的单克隆抗体及其在马铃薯病毒检测上的应用.马铃薯杂志,1992,6(4):233～236
[21]孟清,张鹤龄,宋伯符.应用Dot-ELISA检测PVX、PVY和PVS.中国病毒学,1993,8(4):355～371
[22]张国柱.马铃薯病毒检测技术NCM-ELISA的应用.山西农业科学,1991,(4):25～26
[23]刘卫平.快速ELISA法鉴定马铃薯病毒.马铃薯杂志,1997,11(1):11～12
[24]时杰,E.E.Banttri.用Ig指示试纸法同时检测多种马铃薯病毒.马铃薯杂志,1998,4(4):352～357
[25]李广存，王秀丽，杨辉等.马铃薯病毒检测中DAS-ELISA的改进及注意问题.中国马铃薯，2001,15(5):305-307
[26]韦石泉.植物病毒的免疫电子显微技术.沈阳农业大学学报,1987,18(1):69～75
[27] 刘勇，杨树军，李飞天.应用斑点免疫结合法和组织印迹法检测烟草组织中的TMV和CMV.烟草科技，2000，12：42-44
[28]洪霓，王国平.苹果褪绿叶斑病毒提纯及抗血清制备技术研究,中国果树，1999（1）：15-18
[29]李浩弋,吴元华,赵秀香等.马铃薯Ｙ病毒的RT-PCR检测.沈阳农业大学学报,1999,30(3):244～246
[30]王明霞.应用PCR微量板杂交法植物病毒和类病毒.植物保护,1996,22(1):34～35
[31]侯义龙.果树主要病毒RT-PCR检测体系的建立、优化及病毒特异DNA片段克隆测序研究.[博士学位论文].沈阳:沈阳农业大学,2000.6
[32]周雪平,濮祖芹.应用斑点法检测植物病毒的研究.病毒学杂志,1990(3):317～320
[33]钱秀红,周雪平.斑点免疫测定法在植物病毒研究中的应用及技术要点.生物技术,1994,4(4):39～41
[34]陈剑平,阮义理.电印迹免疫分析(EBIA)及其在植物病毒检测和鉴定中的应用.植物检疫,1989,3(1):24～28
[35]刘品贤.一种新型病毒检测技术.植物医生,1995(4):39
[36]张建军,谢为龙.兰花病毒病研究进展.植物检疫,1999,13(2):109～111
[37]谢为龙,谭群英.加强兰花的病毒检疫.拱北动植物检疫局文集(1990—1994).162～164
[38]卢圣栋.现代分子生物学实验技术.第2版.北京:中国协和医科大学出版社,1999.195～220
[39]王明霞,陈菁瑛.两种非放射性DNA探针检测菊花矮化类病毒的比较.福建省农科院学报,1997,12(1):56～59
[40]邱文平.植物病毒的分子生物学检测方法.热带作物研究,1992(2):65～69
[41]于绍夫.落叶果树的主要病毒病害.北京:农业出版社,1993.80
[42]吴乃虎.基因工程原理.第2版.北京:科学出版社,2000.100
[43]梁国栋.最新分子生物学实验技术.北京:科学出版社,2001.129
[44]穆里斯KB,费里Ｆ,吉布斯Ｒ,等.聚合酶链式反应.北京:科学出版社,1997.93
[45]孔宝华,陈海如RT-PCR检测李坏死环斑病毒的研究.植物检疫,2000,14(5):257～260
[46]潘俊松,刘志昕.建兰花叶病毒外壳蛋白基因序列分析及病毒检测.农业生物技术学报,1999,7(4):325～328
[47]吴淑华,王朝辉.江苏省玉米粗缩病病原病毒的RT-PCR检测.农业生物技术学报,2000,8(4):369～372
[48]陈海如,刘进元.99昆明世博会植物检疫病毒快速检测体系已建立.植物检疫,1999,13(4):239
[49]辛忠涛,李君文,王新为等.一步多重PCR同时检测甲型肝炎和脊髓灰质炎病毒研究. 微生物学免疫学进展，2000，28（4）：43-46
[50]罗瑞梧等.山东省大豆花叶病毒株系鉴定,山东农业科学,1990,5:16～19
[51]陈剑平等.胶体金免疫电镜技术检测和鉴定病汁液中不同形态的植物病毒.植物病理学报,1993,23(2):169～174
[52]徐均焕等.用Ａ蛋白免疫电镜快速检测几种蔬菜作物的两种杆状病毒.植物病理学报,1996,26(3):231～236
[53]成卓敏,等.应用聚合酶链式反应方法检测携带大麦黄矮病毒的单头蚜虫。病毒学报,1992,8(1):80～84
[54]相宁,等.RT-PCR检测烟草环斑病毒的研究.植物检疫,1995,6:337～339
[55]单卫星,等.PCR技术在植物病原物和植物抗病研究中的应用.植物生理学通讯,1995,77(2):137～143
[56]朱作为,等.甘薯羽状班驳病毒分离提纯及血清学特性.1993,8(1):84～88
[57]蔡伟和,洪益国,黄福新.中国南瓜曲叶双生病毒的生物学、血清学和分子杂交的研究〔J〕.中国病毒学报,1994(3):222-224.
[58]张建军.一种快速简便的植物病毒检测方法〔J〕.植物检疫,1998(4):222-223.
[59]尚佑芬.国内外植物病毒研究发展根况〔J〕.山东农业科学,1998(1):51-53.
[60]王海河,高文臣,魏宁生.渭北烟区黄瓜叶病毒酶联免疫检测方法〔J〕.西北农业大学学报1999(4):7-11.
[61]胡向武,陈士超,张林普.黄瓜花叶病毒的侵染扩散及病变细胞的电镜观察〔J〕.安徽师范大学学报,1998(4):354-357.
[62]徐平东,李梅.免疫胶体金技术及其在植物病毒研究中的应用〔J〕.植物检疫,1993(1):26-28.
[63]肖荣堂.聚合酶链反应及其在植物病毒鉴定中的应用〔J〕.植物检疫,1993(4):320-322.
[64]李向东,李怀方，范在丰.马铃薯Y病毒属病毒的分子生物学研究进展.微生物学通报,1999,26(4)283-285
[65]袁小环，李青.血清学方法和分子生物学方法检测植物病毒研究进展.热带农业科学，2001,6：63-68
[66]吴志明，朱水芳等，应用RT-PCR法快速检测马铃薯卷叶病毒，河北农业大学学报，2000，4：77-79
[67]周莉娟 郑伟文等.PCR法检测香蕉囊顶病毒，福建农业学报，2001，4：45-48
[68]贾力，吴茂森等.大麦黄矮病毒单、双价外壳蛋白基因植物表达载体构建及小麦遗传转化，农业生物技术学报，2001，1：23-27
[69]朱常香，宋云枝等.抗芜菁花叶病毒转基因大白菜的培育，植物病理学报，2001，3：257-264
[70]黄贵修，吴坤鑫等.利用mRNA差别显示技术分离橡胶树死皮病相关cDNA，热带作物学报，2002，3，36-42
[71]李彬，周明华等，烟草花叶病毒斑点免疫诊断试剂盒的研制，江苏农业学报，2001，2：91-94
[72]郭兴启，吕士恩，朱汉城等.烟草上马铃薯Y病毒的提纯与检测，中国烟草科学，1999，2：5-7
[73]饶雪琴，蓝翠钰.广州市郊及其邻近地区辣椒CMV和TMV的鉴定，江西农业大学学报，2003，4：558-561
[74]蒋军喜，陈正贤等.我国12省市玉米矮花叶病毒病原鉴定及病毒致病性测定，植物病理学报，2003，4：307-312
[75]李义强，王维超等，豫中烟区烟草病毒病种类鉴定及对策分析，中国烟草科学，2003，1：40-42
[76]尚巧霞，韩成贵等.小麦矮花叶病毒的血清学检测，中国农学院学报.2002，2：15-17
[77]吉容，崔治中等，Ⅰ型马立克氏病毒特异性核酸探针试剂盒的制备盒应用，中国预防兽医学报，2002，4：263-266
[78]史成银，黄捷等.常温核酸探针点杂交检测对虾白斑综合症病毒的PCR扩增产物，农业生物技术学报，2003，2：214-215
[79]吴兴泉，陈士华等.分子生物学技术在马铃薯病毒检测中的应用，中国马铃薯，2003，3：175-179
[80]邓永超，姜涛，范宝昌.应用一步法和套式PCR法检测SARS患者标本中的SARS病毒核酸序列,军事医学科学院院刊.2003,3:166-168
[81]袁青，殷幼平等.马铃薯病毒病分子检测技术研究进展，中国马铃薯，2003，1：33-36
[82]陈南海,杨立,裘维蕃．应用DIBA检测植物病毒的研究[J]．植物病理学报，1992，22（10）：89-93
[83]徐东东，刘永清，王国平.柑橘衰退病毒研究进展,果树学报，2003,20(3):224-229
[84]蒋军喜，周雪平，洪剑.杭州地区甘蔗花叶病病原鉴定，农业生物技术学报，2002，10（4）：377-380
[85]胡建和，王天有等.RT-PCR和nested-PCR技术在猪瘟病毒检测中的应用，甘肃畜牧兽医.2003，1：2-4
[86]李瑞芳，李立等.用套式PCR检测法式囊中的法式囊病病毒，预防兽医学进展，1999，3：50-51
[87]陈君帜，彭辉银等，昆虫质多角体病毒研究的若干新近展，昆虫学报.2002，6：815-821
[88]陈燕芳，陈京，宋淑敏，李明福. 烟草环斑病毒的检疫检测方法，植物检疫，1999，13（4）：215-217
[89]胡伟贞,由雪娟.免疫电镜技术在植物病毒研究中的应用和进展[J].植物检疫,1990,4(5):356 359.
[90]魏梅生.斑点免疫金和免疫金/银染色法检测烟草环斑病毒[J].植物检疫,2000,14(1):1 2.
[91]王劲波，王凤龙，时焦.山东省烟草病毒病毒原鉴定，中国烟草科学1998,(1):26—29
[92]侯明生，赵明富，覃瑞.湖北省白肋烟病毒病毒源种类鉴定，华中农业大学学报，1998，17（6）527-530
[93]王秀芳,朱常香,温孚江等.一个马铃薯Ｙ病毒山东分离物的分离与鉴定，植物病理学报2003,33(3):203-208
[94]陈顺辉，顾钢，巫升鑫等.福建省烟草病毒病种类的检测与防治，烟草科技，2003，9：42-46
[95]张俊华，张明厚等.黑龙江省烟草病毒病流行特征分析，东北农业大学学报，2002，3：209-212
[96]徐修礼，薛彩芳.双抗体夹心ELISA定量检测内毒素地方法研究，细胞与分子免疫学杂志，2002，2:27-30
[97]马书尚，阮小凤,杨勇.苹果褪绿叶斑病毒的ELISA检测技术研究，西北农业大学学报，2000，2：27-30
[98]魏梅生，范晓红.南方菜豆花叶病毒酶联试剂盒的测评，植物检疫，1999，5：281-283
[99]范国成，周乐峰.应用酶联免疫吸附测定技术检测香蕉囊顶病毒，福建农业科学，1999，1：19-22
[100]梁训生等,植物病毒血清学技术.农业出版社,1985
[101]Abdul Samad N,Ari Z,The use of antibody-sensitized latex to detect Cymbidium mosaic virus in orchids.Pertemika Journal of Tropicl Agriculture Science,1993,16(2)：157-160
[102]Adib Rowhani,Chay C,Golino D A,etal.Development of a PCR technique for the detection of grapevine fan leaf virus using grapevine tissue[J]Phytopathology,1993，87（7）749-753
[103]Borja M J,Ponz F.An appraisal of different methods for the detection of the walnut strain of cherry leaf roll virus.Journal of Virological Methods,1992(36):78-83
[104]Boulila M.Prune dwarf ilarvirus in Tunisia detection by biological,serological and electromicroscopic methods and biological characterization of analmond isolate using arange of herbaceous hosts in Tunisia[J],Bulletin OEPP,2002,32(3)：515-519
[105]Cadena-H inojosa M A & R N Campbell. Serological detection of feathery mottle virus strains in sweet potato and Ipomoea incarnata .Plant Dis.1981,(65):412～414
[106]Cadena-Hinojosa M.A.Characterization of isolates of four aphid-trandmitted sweet potato virus.Phytopathology,1981,71:1086～1089
[107]CaliB B & J W Moyer. Purification,serology and particle morphology of two russet crack
strains of sweet potato feathery mottle virus.Photopathol.1981,(71):302～305
[108]Candress T.Recent developments in plant viruses detection[J].Acta Horticulturae, 1995,386:601-605
[109]Candresse T.An immunocapture PCR assay adapted to the detection and the analysis of the molecular variability of apple chlorotic leaf spot virus[J].Acta Horticulturae,1995,386:136-147
[110]Crossley S J.IC-PCR amplification of apple stem grooving virus isolates and comparison of polymerase and coat protein gene sequences[J].Acta Hort,1998,472:113-117
[111]Dodds J A,Jarupat T,Roistacher C N,etal.Detection of strain specific double-stranded RNAs in Citus species infected with citrus trissteza virus:a review[J].Phytophylactica,1987,19:131- 137
[112]Edwards M L.etal.Plant virus detection using a new form of indirect ELISA.Virological Methods,1985,11:309-319
[113]Elliott-MS,etal.Problems with interpretation of serological assays in a virus survey of orchid species from Puerto Rico,Ecuador,and Florida.Plant Disease,1996，80(10)：1160-1164
[114]Esmenjaud D,Abda P.Detection of a region of the coat protein gene of grapevine fanleaf virus by RT-PCR in the nematode vector xiphinema index [J].Plant Disease,1994,78(11)：1087-1090
[115]Fitchc M M M,Lehrer A T,Kormor E,etal.Elimination of sugar cane yellow leaf virus from infected sugar cane plants by meristem tip culture visualized by tissue blot immuo assay[J].Plant Pathology,2001,50(6)：676-680
[116]Fuchs M.Pinc M,Etienne L,etal.Characterization and detection of grapevine fanleaf virus by using cDNA probles[J]Phytopathology,1991,81(5:) 559-565
[117]Hilf M E,Karasev A V,Albiach-Marti M R.Two paths of sequence divergence in the citrus tristeza virus complex[J],Phytopathology,1999,89:336-342
[118]Hu J S,Li H P,Barry K,etal.Comparison of dot blot,ELISA,and RT-PCR assays for detection of two cucumber mosaic virus isolates infecting banana in Hawaii,Plant Disease, 1995 (79): 902-906
[119]Hyun Ryu K,Yoon K E,Wok P.Detection by RT-PCR of Cymbidium mosaic virus in Orchids,Journal of Phytopathology,1995(143):643-646
[120]Inoaye S,Hondo R,Microplate hybridization of amplified vurial DNA segment,J Clin Microbiol,1990,28:1469-1472
[121]James D,Jelkmann W,Upton C.Specific detection of cherry mottle leaf virus using digoxigenin-labeled cDNA probes and RT-PCR.Plant Disease ,1999,83(3):235-239
[122]Janes Rac Torranle L.Development and Application in virus testing.1985 178-183
[123]Jawee A,Adama A N Serological detection of strawberry mild yellow edge as sociated virus[J].Acta Horticulturae，1995，385：98-105
[124]Jiao K,Zhang S S,Sun W.Sensitive detection of a plant virus by electrochemical Enzyme linked immunoassay[J]Fresenius’Journal Analytical chemistry,2000,367(7)：667-671
[125]Kadenkrenziger D,Lamprecht S,Martin R R.IC-PCR assay and ELISA for the detection of strawberry mild yellow edge association potexvirus[J].Acta Horticulturae,1995,385:33-38
[126]Karasev M C,Dawson W O,Hilf M E, etal.Molecule biology of citrus tristeza virus :implication for disease diagnosis and control[J].Acta Horticulturuae,1998,472:333-337
[127]Kinard G R,Scott S W,Barnett O W.Detection of apple chlorotic leaf spot and apple stem growing viruses using RT-PCR.Plant Disease,1996,80(6):616-621
[128]Kinard G R.Detection of apple chlorotic leaf spot and apple stem grooving viruses using
RT-PCR[J].Plant Disease,1996,80(6):616-622
[129]Kohler G ,Milstein C. Continuous culoures of fused cells secreting antibody of predefined specificity Nature,1975,256:490-496
[130]Koler G,Milstein C.Continuous cultures of fuesd cells secreting antibody of predefined specificity.Nature,1975,256:494-496
[131]Kummert J.Sensitive detection of apple stem grooving and apple stem pitting viruses from infected apple trees by RT-PCR[J].Acta Hort,1998，472：97-104
[132]M.H.V.Van Regen mortal serology and immunochememistry 1982 113-121
[133]M.Singh,R.P.Singh,L.Moore.Evaluation of NASH and RT-PCR for the Detection of PVY in the Dormant Tubers and Its Comparison With Visual Symptoms and ELISA in Plant,Amer J of Potato Res,76(2)：61-66
[134]Malinowski T,Detection of apple stem pitting virus and pear vein yellows virus using reverse transcription polymetase chain reaction[J]. Acta Horticulturae,1998,472:87-95
[135]Minafra A,Hadidi A,Martelloi G P.Detection of grapevine clostero virus A in infected grapevine tissue by RT-PCR[J].Vitis,1992(31):221-227
[136]Mink G I,Aichele M D.Detection of prunue necrotic ring spot and prune dwarf viruses in prunus seed and seedling by ELISA[J]Plant Disease,1984,68(5)：378-381
[137]Moreno P,Guerri J,Munoz N.Identification of Spanish strais of citrus tristeza virus by analysis of double-stranded RNA[J].Phytopathology,1990,80:477-482
[138]Mullis K,etal. specific enzymatic amplification of DNA in vitro:The polymerase chain reaction.Cold Sping Harbor Symp.Quant.Biol.,1986,51:263
[139]Nemchinov L.,Hadidi A.PCR-detection of apple stem pitting virus from pome fruit hosts and sequence variability among viral isolates[J].Acta Hort,1998,472:67-69
[140]Nemchinov L.Sensititive detection of apple chlorotic leaf spot virus from infected apple or peach tissue using RT-PCR IC-RT-PCR or multiplex IC-RT-PCR[J].Acta Horticulurae,1995,386:51-57
[141]P.Ellis,R.Stace Smith,G.de.Villers.Identification and Geographic Doistribution of Potato Virus Y,Plant disease,1997,81(5)：481-484
[142]Parakh D B,Shamioul A M,Hadidi A,etal.Detection of prune dwarf ilarvirus from infected stone fruits using reverse transcription –polymerase chain reaction.Acta Horticulture,1995 (386): 421-430
[143]Parakh D R,Shamloul A M,Hadidi A,etal.Detection of prune dwarf ilarvirus from infected stone fruits using RT-PCR[J].Acta Horticulturae,1995,386:421-430.
[144]Rowhan A,Maningas M A,Lile L S，etal.Development of a detection system for viruses of woody plants based on PCR analysis of immobilized virions,Phytopathology,1995,85(3):347-352
[145]Rudra P. etal Development of the molecular methods for potato virus and viroid detection and prevention. Genome,1992,5:89-93
[146]Saade M,Aparicio F,Sanchez Navarro J A,etal.Simultaneous detection of the three ilarviruses affecting stone fruit trees by nonisotopic molecular hybridization and multiple reverse transcription polymerase chain reaction[J],Phytopathology，2000，90（12）：1330-1336
[147]Saldarelli P,Minafra A,Martelli G P,etal.Detection of grapevine leaf roll associated clostero virusⅢ by molecular hybridization[J].Plant Pathology,1994,43:91-96
[148]Spiegel S,Scott S W,Bowman-Vance V,etal.Improved detection of prunus necrotic ringspot
virus by the polymeras chain reaction.European Journal of Planrt Pathology,1996(102)：681-685
[149]Tatsuji Hataya.Alice Kazuko Inoue.etal.A PCR-microplate hybridization method for plant virus detection,Journal of Virological Methods.1994,46:223-236
[150]Tet-Fatt Chia,Yang-Sun Chan,Nan-Nai Chua.Characterization of Cymbidium mosaic virus coat protein gene and explanation in transgenic tobacco plants,Plant Molecular Biology, 1992, 18:1091-1099
[151]Vera L A Marinho.Detection of apple stem grooving virus in dormant apple trees with crude extracts a stem plates for One-step RT-PCR[J].Plant Disease ,1996，82（7）：785-790
[152]Vunsh R,etal.The use of the polymerase chain reaction (PCR) for the detection of bean yellow mosaic virus in gladiolus.Ann.Appl.Biol.1990,117:561-569
[153]Watkins C A，Mayo M A,etal.Double stranded RNA analysis of strawberry plants affected by June Yellows [J].Ann..appl.Biol.,1990,116:73-83
[154]Wetzel T,Candresse T.A highly sensitive immunocapture polymerase chain reaction for plum pox potyvirus detection.Journal of virology Methods,1992(39):27-37
[155]Wetzel T,Candresse T.A polymerase chain reaction assay adapted to plum pox potyvirus detection.Journal of Virological methods,1991(33):355-365
[156]Yu L,Li Y,Gu G,etal.A fast,sensitive and specific method for rice dwarf virus detection by Northern blot hybridization [J].Acta Virologica，1999,43(6)：361-365
[2]郭兴启，朱汉城，吕士恩等．酶标记羊抗兔间接法检测烟草马铃薯Y病毒的研究[J]．中国烟草学报，1998，4(2)：38–42．
[3]周本国，高正良，刘小平．安徽省烟草脉斑病(PVY)的发生趋势及防治措施[J]．安徽农学通报，1998，4(1)：34–35．
[4]程宝玉，吴娟霞，陈卫华等．豫西烟草脉斑病发生规律及药剂防治研究[J]．烟草科技，2002，(7)：46–48．
[5]田波，裴美云．植物病毒研究方法[M]．北京：科学出版社，1987，42–43．
[6]王劲波，王凤龙，钱玉梅等．山东烟区主要病毒的株系鉴定[J]．中国烟草学报，1998，4(1)：24–30．
[7]谈文，蒋士君，刘骏等．烟草病理学教程[M]．北京：中国科学技术出版社，1995，129–131．
[8]董继超,李花婷,苏永士,等.河南省烟草主要病害流行规律分析[Ｊ].河南农业大学学报,1992,(26增刊):31-39.
[9]蒋金炜,蒋士君,董继超.烟草病虫害防治[Ｍ].郑州:河南科学技术出版社,1994.87-88.
[10]谈文.烟草病虫害防治手册[Ｍ].北京:金盾出版社,2000.116-118.
[11]马国胜,郭红,高正良,等.固镇县烟草脉斑病流行规律调查研究[Ｊ].烟草科技,2000,(2):46-47.
[12]李嵩震,马中仁,侯文华.河南烟草栽培[Ｍ].郑州:河南科学技术出版社,1997.61-65.
[14]彭宏梅，李月秋，张仲凯等.应用电镜技术研究大理州烟草苗期病毒病的发生状况，云南农业大学学报.2000，15（1）：70-71
[13]周艳玲，刘学敏，孟玉芹.马铃薯Y病毒的检测技术[J].中国马铃薯，2000.14(2)：89-93
[15]Ｅ.库尔斯塔克.植物病毒比较诊断指南,农业出版社,1991,518
[16]王秀芳，任广伟，王凤龙.烟草上马铃薯Y病毒的RT-PCR检测，中国烟草学报,2003，9(2)28-30
[17]吴红芝，孔宝华，陈海如等.昆明地区菊花病毒病的调查与鉴定. 云南农业大学学报，2002，17（1），24-27
[18]张鹤龄.酶联免疫吸附测定(ELISA)及其在植物病毒鉴定中的应用.马铃薯,1983,(4):35～41
[19]仲乃琴.ELISA技术检测马铃薯病毒的研究，甘肃农业大学学报，1998，33（2）：178-181
[20]李汝刚,郭军,鞠振林等.马铃薯Ｘ、Ｙ病毒的单克隆抗体及其在马铃薯病毒检测上的应用.马铃薯杂志,1992,6(4):233～236
[21]孟清,张鹤龄,宋伯符.应用Dot-ELISA检测PVX、PVY和PVS.中国病毒学,1993,8(4):355～371
[22]张国柱.马铃薯病毒检测技术NCM-ELISA的应用.山西农业科学,1991,(4):25～26
[23]刘卫平.快速ELISA法鉴定马铃薯病毒.马铃薯杂志,1997,11(1):11～12
[24]时杰,E.E.Banttri.用Ig指示试纸法同时检测多种马铃薯病毒.马铃薯杂志,1998,4(4):352～357
[25]李广存，王秀丽，杨辉等.马铃薯病毒检测中DAS-ELISA的改进及注意问题.中国马铃薯，2001,15(5):305-307
[26]韦石泉.植物病毒的免疫电子显微技术.沈阳农业大学学报,1987,18(1):69～75
[27] 刘勇，杨树军，李飞天.应用斑点免疫结合法和组织印迹法检测烟草组织中的TMV和CMV.烟草科技，2000，12：42-44
[28]洪霓，王国平.苹果褪绿叶斑病毒提纯及抗血清制备技术研究,中国果树，1999（1）：15-18
[29]李浩弋,吴元华,赵秀香等.马铃薯Ｙ病毒的RT-PCR检测.沈阳农业大学学报,1999,30(3):244～246
[30]王明霞.应用PCR微量板杂交法植物病毒和类病毒.植物保护,1996,22(1):34～35
[31]侯义龙.果树主要病毒RT-PCR检测体系的建立、优化及病毒特异DNA片段克隆测序研究.[博士学位论文].沈阳:沈阳农业大学,2000.6
[32]周雪平,濮祖芹.应用斑点法检测植物病毒的研究.病毒学杂志,1990(3):317～320
[33]钱秀红,周雪平.斑点免疫测定法在植物病毒研究中的应用及技术要点.生物技术,1994,4(4):39～41
[34]陈剑平,阮义理.电印迹免疫分析(EBIA)及其在植物病毒检测和鉴定中的应用.植物检疫,1989,3(1):24～28
[35]刘品贤.一种新型病毒检测技术.植物医生,1995(4):39
[36]张建军,谢为龙.兰花病毒病研究进展.植物检疫,1999,13(2):109～111
[37]谢为龙,谭群英.加强兰花的病毒检疫.拱北动植物检疫局文集(1990—1994).162～164
[38]卢圣栋.现代分子生物学实验技术.第2版.北京:中国协和医科大学出版社,1999.195～220
[39]王明霞,陈菁瑛.两种非放射性DNA探针检测菊花矮化类病毒的比较.福建省农科院学报,1997,12(1):56～59
[40]邱文平.植物病毒的分子生物学检测方法.热带作物研究,1992(2):65～69
[41]于绍夫.落叶果树的主要病毒病害.北京:农业出版社,1993.80
[42]吴乃虎.基因工程原理.第2版.北京:科学出版社,2000.100
[43]梁国栋.最新分子生物学实验技术.北京:科学出版社,2001.129
[44]穆里斯KB,费里Ｆ,吉布斯Ｒ,等.聚合酶链式反应.北京:科学出版社,1997.93
[45]孔宝华,陈海如RT-PCR检测李坏死环斑病毒的研究.植物检疫,2000,14(5):257～260
[46]潘俊松,刘志昕.建兰花叶病毒外壳蛋白基因序列分析及病毒检测.农业生物技术学报,1999,7(4):325～328
[47]吴淑华,王朝辉.江苏省玉米粗缩病病原病毒的RT-PCR检测.农业生物技术学报,2000,8(4):369～372
[48]陈海如,刘进元.99昆明世博会植物检疫病毒快速检测体系已建立.植物检疫,1999,13(4):239
[49]辛忠涛,李君文,王新为等.一步多重PCR同时检测甲型肝炎和脊髓灰质炎病毒研究. 微生物学免疫学进展，2000，28（4）：43-46
[50]罗瑞梧等.山东省大豆花叶病毒株系鉴定,山东农业科学,1990,5:16～19
[51]陈剑平等.胶体金免疫电镜技术检测和鉴定病汁液中不同形态的植物病毒.植物病理学报,1993,23(2):169～174
[52]徐均焕等.用Ａ蛋白免疫电镜快速检测几种蔬菜作物的两种杆状病毒.植物病理学报,1996,26(3):231～236
[53]成卓敏,等.应用聚合酶链式反应方法检测携带大麦黄矮病毒的单头蚜虫。病毒学报,1992,8(1):80～84
[54]相宁,等.RT-PCR检测烟草环斑病毒的研究.植物检疫,1995,6:337～339
[55]单卫星,等.PCR技术在植物病原物和植物抗病研究中的应用.植物生理学通讯,1995,77(2):137～143
[56]朱作为,等.甘薯羽状班驳病毒分离提纯及血清学特性.1993,8(1):84～88
[57]蔡伟和,洪益国,黄福新.中国南瓜曲叶双生病毒的生物学、血清学和分子杂交的研究〔J〕.中国病毒学报,1994(3):222-224.
[58]张建军.一种快速简便的植物病毒检测方法〔J〕.植物检疫,1998(4):222-223.
[59]尚佑芬.国内外植物病毒研究发展根况〔J〕.山东农业科学,1998(1):51-53.
[60]王海河,高文臣,魏宁生.渭北烟区黄瓜叶病毒酶联免疫检测方法〔J〕.西北农业大学学报1999(4):7-11.
[61]胡向武,陈士超,张林普.黄瓜花叶病毒的侵染扩散及病变细胞的电镜观察〔J〕.安徽师范大学学报,1998(4):354-357.
[62]徐平东,李梅.免疫胶体金技术及其在植物病毒研究中的应用〔J〕.植物检疫,1993(1):26-28.
[63]肖荣堂.聚合酶链反应及其在植物病毒鉴定中的应用〔J〕.植物检疫,1993(4):320-322.
[64]李向东,李怀方，范在丰.马铃薯Y病毒属病毒的分子生物学研究进展.微生物学通报,1999,26(4)283-285
[65]袁小环，李青.血清学方法和分子生物学方法检测植物病毒研究进展.热带农业科学，2001,6：63-68
[66]吴志明，朱水芳等，应用RT-PCR法快速检测马铃薯卷叶病毒，河北农业大学学报，2000，4：77-79
[67]周莉娟 郑伟文等.PCR法检测香蕉囊顶病毒，福建农业学报，2001，4：45-48
[68]贾力，吴茂森等.大麦黄矮病毒单、双价外壳蛋白基因植物表达载体构建及小麦遗传转化，农业生物技术学报，2001，1：23-27
[69]朱常香，宋云枝等.抗芜菁花叶病毒转基因大白菜的培育，植物病理学报，2001，3：257-264
[70]黄贵修，吴坤鑫等.利用mRNA差别显示技术分离橡胶树死皮病相关cDNA，热带作物学报，2002，3，36-42
[71]李彬，周明华等，烟草花叶病毒斑点免疫诊断试剂盒的研制，江苏农业学报，2001，2：91-94
[72]郭兴启，吕士恩，朱汉城等.烟草上马铃薯Y病毒的提纯与检测，中国烟草科学，1999，2：5-7
[73]饶雪琴，蓝翠钰.广州市郊及其邻近地区辣椒CMV和TMV的鉴定，江西农业大学学报，2003，4：558-561
[74]蒋军喜，陈正贤等.我国12省市玉米矮花叶病毒病原鉴定及病毒致病性测定，植物病理学报，2003，4：307-312
[75]李义强，王维超等，豫中烟区烟草病毒病种类鉴定及对策分析，中国烟草科学，2003，1：40-42
[76]尚巧霞，韩成贵等.小麦矮花叶病毒的血清学检测，中国农学院学报.2002，2：15-17
[77]吉容，崔治中等，Ⅰ型马立克氏病毒特异性核酸探针试剂盒的制备盒应用，中国预防兽医学报，2002，4：263-266
[78]史成银，黄捷等.常温核酸探针点杂交检测对虾白斑综合症病毒的PCR扩增产物，农业生物技术学报，2003，2：214-215
[79]吴兴泉，陈士华等.分子生物学技术在马铃薯病毒检测中的应用，中国马铃薯，2003，3：175-179
[80]邓永超，姜涛，范宝昌.应用一步法和套式PCR法检测SARS患者标本中的SARS病毒核酸序列,军事医学科学院院刊.2003,3:166-168
[81]袁青，殷幼平等.马铃薯病毒病分子检测技术研究进展，中国马铃薯，2003，1：33-36
[82]陈南海,杨立,裘维蕃．应用DIBA检测植物病毒的研究[J]．植物病理学报，1992，22（10）：89-93
[83]徐东东，刘永清，王国平.柑橘衰退病毒研究进展,果树学报，2003,20(3):224-229
[84]蒋军喜，周雪平，洪剑.杭州地区甘蔗花叶病病原鉴定，农业生物技术学报，2002，10（4）：377-380
[85]胡建和，王天有等.RT-PCR和nested-PCR技术在猪瘟病毒检测中的应用，甘肃畜牧兽医.2003，1：2-4
[86]李瑞芳，李立等.用套式PCR检测法式囊中的法式囊病病毒，预防兽医学进展，1999，3：50-51
[87]陈君帜，彭辉银等，昆虫质多角体病毒研究的若干新近展，昆虫学报.2002，6：815-821
[88]陈燕芳，陈京，宋淑敏，李明福. 烟草环斑病毒的检疫检测方法，植物检疫，1999，13（4）：215-217
[89]胡伟贞,由雪娟.免疫电镜技术在植物病毒研究中的应用和进展[J].植物检疫,1990,4(5):356 359.
[90]魏梅生.斑点免疫金和免疫金/银染色法检测烟草环斑病毒[J].植物检疫,2000,14(1):1 2.
[91]王劲波，王凤龙，时焦.山东省烟草病毒病毒原鉴定，中国烟草科学1998,(1):26—29
[92]侯明生，赵明富，覃瑞.湖北省白肋烟病毒病毒源种类鉴定，华中农业大学学报，1998，17（6）527-530
[93]王秀芳,朱常香,温孚江等.一个马铃薯Ｙ病毒山东分离物的分离与鉴定，植物病理学报2003,33(3):203-208
[94]陈顺辉，顾钢，巫升鑫等.福建省烟草病毒病种类的检测与防治，烟草科技，2003，9：42-46
[95]张俊华，张明厚等.黑龙江省烟草病毒病流行特征分析，东北农业大学学报，2002，3：209-212
[96]徐修礼，薛彩芳.双抗体夹心ELISA定量检测内毒素地方法研究，细胞与分子免疫学杂志，2002，2:27-30
[97]马书尚，阮小凤,杨勇.苹果褪绿叶斑病毒的ELISA检测技术研究，西北农业大学学报，2000，2：27-30
[98]魏梅生，范晓红.南方菜豆花叶病毒酶联试剂盒的测评，植物检疫，1999，5：281-283
[99]范国成，周乐峰.应用酶联免疫吸附测定技术检测香蕉囊顶病毒，福建农业科学，1999，1：19-22
[100]梁训生等,植物病毒血清学技术.农业出版社,1985
[101]Abdul Samad N,Ari Z,The use of antibody-sensitized latex to detect Cymbidium mosaic virus in orchids.Pertemika Journal of Tropicl Agriculture Science,1993,16(2)：157-160
[102]Adib Rowhani,Chay C,Golino D A,etal.Development of a PCR technique for the detection of grapevine fan leaf virus using grapevine tissue[J]Phytopathology,1993，87（7）749-753
[103]Borja M J,Ponz F.An appraisal of different methods for the detection of the walnut strain of cherry leaf roll virus.Journal of Virological Methods,1992(36):78-83
[104]Boulila M.Prune dwarf ilarvirus in Tunisia detection by biological,serological and electromicroscopic methods and biological characterization of analmond isolate using arange of herbaceous hosts in Tunisia[J],Bulletin OEPP,2002,32(3)：515-519
[105]Cadena-H inojosa M A & R N Campbell. Serological detection of feathery mottle virus strains in sweet potato and Ipomoea incarnata .Plant Dis.1981,(65):412～414
[106]Cadena-Hinojosa M.A.Characterization of isolates of four aphid-trandmitted sweet potato virus.Phytopathology,1981,71:1086～1089
[107]CaliB B & J W Moyer. Purification,serology and particle morphology of two russet crack
strains of sweet potato feathery mottle virus.Photopathol.1981,(71):302～305
[108]Candress T.Recent developments in plant viruses detection[J].Acta Horticulturae, 1995,386:601-605
[109]Candresse T.An immunocapture PCR assay adapted to the detection and the analysis of the molecular variability of apple chlorotic leaf spot virus[J].Acta Horticulturae,1995,386:136-147
[110]Crossley S J.IC-PCR amplification of apple stem grooving virus isolates and comparison of polymerase and coat protein gene sequences[J].Acta Hort,1998,472:113-117
[111]Dodds J A,Jarupat T,Roistacher C N,etal.Detection of strain specific double-stranded RNAs in Citus species infected with citrus trissteza virus:a review[J].Phytophylactica,1987,19:131- 137
[112]Edwards M L.etal.Plant virus detection using a new form of indirect ELISA.Virological Methods,1985,11:309-319
[113]Elliott-MS,etal.Problems with interpretation of serological assays in a virus survey of orchid species from Puerto Rico,Ecuador,and Florida.Plant Disease,1996，80(10)：1160-1164
[114]Esmenjaud D,Abda P.Detection of a region of the coat protein gene of grapevine fanleaf virus by RT-PCR in the nematode vector xiphinema index [J].Plant Disease,1994,78(11)：1087-1090
[115]Fitchc M M M,Lehrer A T,Kormor E,etal.Elimination of sugar cane yellow leaf virus from infected sugar cane plants by meristem tip culture visualized by tissue blot immuo assay[J].Plant Pathology,2001,50(6)：676-680
[116]Fuchs M.Pinc M,Etienne L,etal.Characterization and detection of grapevine fanleaf virus by using cDNA probles[J]Phytopathology,1991,81(5:) 559-565
[117]Hilf M E,Karasev A V,Albiach-Marti M R.Two paths of sequence divergence in the citrus tristeza virus complex[J],Phytopathology,1999,89:336-342
[118]Hu J S,Li H P,Barry K,etal.Comparison of dot blot,ELISA,and RT-PCR assays for detection of two cucumber mosaic virus isolates infecting banana in Hawaii,Plant Disease, 1995 (79): 902-906
[119]Hyun Ryu K,Yoon K E,Wok P.Detection by RT-PCR of Cymbidium mosaic virus in Orchids,Journal of Phytopathology,1995(143):643-646
[120]Inoaye S,Hondo R,Microplate hybridization of amplified vurial DNA segment,J Clin Microbiol,1990,28:1469-1472
[121]James D,Jelkmann W,Upton C.Specific detection of cherry mottle leaf virus using digoxigenin-labeled cDNA probes and RT-PCR.Plant Disease ,1999,83(3):235-239
[122]Janes Rac Torranle L.Development and Application in virus testing.1985 178-183
[123]Jawee A,Adama A N Serological detection of strawberry mild yellow edge as sociated virus[J].Acta Horticulturae，1995，385：98-105
[124]Jiao K,Zhang S S,Sun W.Sensitive detection of a plant virus by electrochemical Enzyme linked immunoassay[J]Fresenius’Journal Analytical chemistry,2000,367(7)：667-671
[125]Kadenkrenziger D,Lamprecht S,Martin R R.IC-PCR assay and ELISA for the detection of strawberry mild yellow edge association potexvirus[J].Acta Horticulturae,1995,385:33-38
[126]Karasev M C,Dawson W O,Hilf M E, etal.Molecule biology of citrus tristeza virus :implication for disease diagnosis and control[J].Acta Horticulturuae,1998,472:333-337
[127]Kinard G R,Scott S W,Barnett O W.Detection of apple chlorotic leaf spot and apple stem growing viruses using RT-PCR.Plant Disease,1996,80(6):616-621
[128]Kinard G R.Detection of apple chlorotic leaf spot and apple stem grooving viruses using
RT-PCR[J].Plant Disease,1996,80(6):616-622
[129]Kohler G ,Milstein C. Continuous culoures of fused cells secreting antibody of predefined specificity Nature,1975,256:490-496
[130]Koler G,Milstein C.Continuous cultures of fuesd cells secreting antibody of predefined specificity.Nature,1975,256:494-496
[131]Kummert J.Sensitive detection of apple stem grooving and apple stem pitting viruses from infected apple trees by RT-PCR[J].Acta Hort,1998，472：97-104
[132]M.H.V.Van Regen mortal serology and immunochememistry 1982 113-121
[133]M.Singh,R.P.Singh,L.Moore.Evaluation of NASH and RT-PCR for the Detection of PVY in the Dormant Tubers and Its Comparison With Visual Symptoms and ELISA in Plant,Amer J of Potato Res,76(2)：61-66
[134]Malinowski T,Detection of apple stem pitting virus and pear vein yellows virus using reverse transcription polymetase chain reaction[J]. Acta Horticulturae,1998,472:87-95
[135]Minafra A,Hadidi A,Martelloi G P.Detection of grapevine clostero virus A in infected grapevine tissue by RT-PCR[J].Vitis,1992(31):221-227
[136]Mink G I,Aichele M D.Detection of prunue necrotic ring spot and prune dwarf viruses in prunus seed and seedling by ELISA[J]Plant Disease,1984,68(5)：378-381
[137]Moreno P,Guerri J,Munoz N.Identification of Spanish strais of citrus tristeza virus by analysis of double-stranded RNA[J].Phytopathology,1990,80:477-482
[138]Mullis K,etal. specific enzymatic amplification of DNA in vitro:The polymerase chain reaction.Cold Sping Harbor Symp.Quant.Biol.,1986,51:263
[139]Nemchinov L.,Hadidi A.PCR-detection of apple stem pitting virus from pome fruit hosts and sequence variability among viral isolates[J].Acta Hort,1998,472:67-69
[140]Nemchinov L.Sensititive detection of apple chlorotic leaf spot virus from infected apple or peach tissue using RT-PCR IC-RT-PCR or multiplex IC-RT-PCR[J].Acta Horticulurae,1995,386:51-57
[141]P.Ellis,R.Stace Smith,G.de.Villers.Identification and Geographic Doistribution of Potato Virus Y,Plant disease,1997,81(5)：481-484
[142]Parakh D B,Shamioul A M,Hadidi A,etal.Detection of prune dwarf ilarvirus from infected stone fruits using reverse transcription –polymerase chain reaction.Acta Horticulture,1995 (386): 421-430
[143]Parakh D R,Shamloul A M,Hadidi A,etal.Detection of prune dwarf ilarvirus from infected stone fruits using RT-PCR[J].Acta Horticulturae,1995,386:421-430.
[144]Rowhan A,Maningas M A,Lile L S，etal.Development of a detection system for viruses of woody plants based on PCR analysis of immobilized virions,Phytopathology,1995,85(3):347-352
[145]Rudra P. etal Development of the molecular methods for potato virus and viroid detection and prevention. Genome,1992,5:89-93
[146]Saade M,Aparicio F,Sanchez Navarro J A,etal.Simultaneous detection of the three ilarviruses affecting stone fruit trees by nonisotopic molecular hybridization and multiple reverse transcription polymerase chain reaction[J],Phytopathology，2000，90（12）：1330-1336
[147]Saldarelli P,Minafra A,Martelli G P,etal.Detection of grapevine leaf roll associated clostero virusⅢ by molecular hybridization[J].Plant Pathology,1994,43:91-96
[148]Spiegel S,Scott S W,Bowman-Vance V,etal.Improved detection of prunus necrotic ringspot
virus by the polymeras chain reaction.European Journal of Planrt Pathology,1996(102)：681-685
[149]Tatsuji Hataya.Alice Kazuko Inoue.etal.A PCR-microplate hybridization method for plant virus detection,Journal of Virological Methods.1994,46:223-236
[150]Tet-Fatt Chia,Yang-Sun Chan,Nan-Nai Chua.Characterization of Cymbidium mosaic virus coat protein gene and explanation in transgenic tobacco plants,Plant Molecular Biology, 1992, 18:1091-1099
[151]Vera L A Marinho.Detection of apple stem grooving virus in dormant apple trees with crude extracts a stem plates for One-step RT-PCR[J].Plant Disease ,1996，82（7）：785-790
[152]Vunsh R,etal.The use of the polymerase chain reaction (PCR) for the detection of bean yellow mosaic virus in gladiolus.Ann.Appl.Biol.1990,117:561-569
[153]Watkins C A，Mayo M A,etal.Double stranded RNA analysis of strawberry plants affected by June Yellows [J].Ann..appl.Biol.,1990,116:73-83
[154]Wetzel T,Candresse T.A highly sensitive immunocapture polymerase chain reaction for plum pox potyvirus detection.Journal of virology Methods,1992(39):27-37
[155]Wetzel T,Candresse T.A polymerase chain reaction assay adapted to plum pox potyvirus detection.Journal of Virological methods,1991(33):355-365
[156]Yu L,Li Y,Gu G,etal.A fast,sensitive and specific method for rice dwarf virus detection by Northern blot hybridization [J].Acta Virologica，1999,43(6)：361-365