3种甜樱桃病毒PNRSV、PDV及LChV-2的多重RT-PCR检测方法的建立与应用

doi:10.3864/j.issn.0578-1752.2014.06.007

摘要/Abstract

摘要： 【目的】建立可同时检测李属坏死环斑病毒（Prunus necrotic ringspot virus，PNRSV）、李矮缩病毒（Prune dwarf virus，PDV）、樱桃小果病毒2（Little cherry virus-2，LChV-2）的多重RT-PCR检测方法。【方法】以复合感染3种病毒的甜樱桃病株叶片为材料，采用CTAB法提取样本总RNA，选用随机六聚体引物对植物样本总RNA进行反转录，所得cDNA作为多重RT-PCR的扩增模板。根据GenBank中PNRSV、PDV、LChV-2基因组序列共设计6对特异引物，分别通过单一RT-PCR和多重RT-PCR筛选出可用于同时检测3种甜樱桃病毒的引物组合。对多重RT-PCR的退火温度及循环数进行优化，以筛选出各引物组合的最适扩增条件。分别以单一感染PNRSV、PDV、LChV-2、复合感染3种病毒、单一感染樱桃病毒A（Cherry virus A，CVA）及甜樱桃无毒苗为样本，对多重RT-PCR引物的特异性进行分析。选取复合感染3种病毒的甜樱桃总RNA的反转录产物为初始模板，按照梯度稀释法依次将模板稀释为2、22、23、24、25倍，在相同PCR反应体系及反应条件下分别对各引物组合的灵敏度进行分析。多重RT-PCR的扩增条带经凝胶回收试剂盒回收纯化后连接至pMD18-T vector，克隆测序，以验证多重RT-PCR检测的准确性。并应用该方法对山东泰安地区甜樱桃生产园中间隔栽培的中国樱桃进行检测。【结果】筛选到2个可以应用的引物组合，组合1“PNRSV-S1/A1、PDV-S2/A2、LChV2-S1/A1”可分别特异性地扩增733、467、337 bp的片段。组合2“PNRSV-S1/A1、PDV-S3/A3、LChV2-S1/A1”可分别扩增得到733、265、337 bp的片段。扩增产物大小与预期相符。多重RT-PCR反应条件优化结果显示，在退火温度52℃、35个循环条件下，2个引物组合的检测效果均较为理想。特异性分析结果显示，2个引物组合均能特异性检测其各自的靶病毒。灵敏度分析结果显示，2个引物组合在cDNA的23×稀释液中仍能特异性扩增，但扩增条带的强度稍有差异，其对植物总RNA的反转录产物的最低检测浓度为107.9 ng•μL-1。克隆测序及序列分析表明，2个引物组合对各自靶病毒的检测结果可靠。应用该方法对9个中国樱桃样本进行检测，结果显示，测试样品均至少感染了2种病毒，其中5个样品复合感染了3种病毒，2个样品同时感染PDV和LChV-2，2个样品同时感染PNRSV和LChV-2。【结论】应用建立的多重RT-PCR检测方法可稳定、准确、灵敏的同时检测单一或复合侵染的3种甜樱桃病毒。

关键词: 李属坏死环斑病毒 , 李矮缩病毒 , 樱桃小果病毒-2 , 多重RT-PCR , 病原检测

Abstract: 【Objective】The objective of this study is to develop a multiplex RT-PCR protocol to detect 3 sweet cherry virus species Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV) and Little cherry virus-2 (LChV-2) simultaneously. 【Method】 The leaves of the sweet cherry (Prunus avium Lindl.) infected by 3 virus species were selected as the experimental materials. Total RNA was extracted using CTAB extraction buffer. For cDNA synthesis, reverse transcription was carried out using random hexamer primer. Six pairs of primers were designed according to the genome sequences of PNRSV, PDV and LChV-2 which were published in GenBank. Single RT-PCR and multiplex RT-PCR were carried out, respectively, to select the primer groups which could be used in the multiplex virus detection. Annealing temperature and the number of the PCR cycles were evaluated to optimize the multiplex RT-PCR conditions. The specificity of the primers was analyzed by using the single-virus infected samples, complex-virus infected samples and virus-free leaf samples. To analyze the sensitivity of the multiples RT-PCR, the transcript which was prepared from the complex-virus infected samples was diluted to 2-fold series. All of the reactions were carried out in the same reaction buffer and under the same conditions. To confirm the accuracy of the multiplex RT-PCR, each amplified fragment was purified by DNA gel extraction kit, cloned into pMD18-T vector and sequenced. In this paper, the multiplex RT-PCR was also used to detect virus infection in Chinese cherry (P. pseudocerasus Lindl.) trees which are cultivated in the orchards in Taian, Shandong Province. 【Result】 Two primer groups were selected and could be used in the multiplex RT-PCR detection. Group 1 including primers ‘PNRSV-S1/A1, PDV-S2/A2 and LChV2-S1/A1’ amplified the fragments of 733, 467 and 337 bp, respectively. Group 2 including ‘PNRSV-S1/A1, PDV-S3/A3, LChV2-S1/A1’ amplified the fragments of 733, 265 and 337 bp, respectively. The PCR products were consistent with the expected size. It was showed that both of the two primer groups could be used to detect 3 sweet cherry virus species with high specificity. Sensitivity analysis showed that virus fragments could be detected in the 23 dilution of the plant cDNA using both of the two primer groups. However, the intensity of each virus fragments was slightly different. The lowest concentration of the plant total RNA transcripts was 107.9 ng•μL-1. Sequence analysis confirmed that it was reliable and accurate to detect the viruses using these two primer groups. This method was then applied to detect the incidence of virus disease in Chinese cherry trees. The results indicated that all of the samples were infected by 2 virus species at least. Of the 9 detected samples, 5 of them were infected by 3 virus species simultaneously. Two of them were infected by PDV and LChV-2 and another two samples were infected by PNRSV and LChV-2. 【Conclusion】 The multiplex RT-PCR protocol can simultaneously detect PNRSV, PDV and LChV-2 with high stability, accuracy and sensitivity.

Key words: Prunus necrotic ringspot virus (PNRSV) , Prune dwarf virus (PDV) , Little cherry virus-2 (LChV-2) , multiplex RT-PCR , pathogen detection

宗晓娟, 王文文, 魏海蓉, 王甲威, 陈新, 徐丽, 刘庆忠. 3种甜樱桃病毒PNRSV、PDV及LChV-2的多重RT-PCR检测方法的建立与应用[J]. 中国农业科学, 2014, 47(6): 1111-1118.

ZONG Xiao-Juan, WANG Wen-Wen, WEI Hai-Rong, WANG Jia-Wei, CHEN Xin, XU Li, LIU Qing-Zhong. Development and Application of a Multiplex RT-PCR Assay for Detecting Three Sweet Cherry Virus Species[J]. Scientia Agricultura Sinica, 2014, 47(6): 1111-1118.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2014.06.007

https://www.chinaagrisci.com/CN/Y2014/V47/I6/1111

参考文献

[1]Myrta A, Savino V. Virus and virus-like diseases of cherry in the Mediterranean region. Acta Horticulturae, 2008, 795: 891-896.

[2]Isogai M, Aoyagi J, Nakagawa M, Kubodera Y, Satoh K, Katoh T, Inamori M, Yamashita K, Yoshikawa N. Molecular detection of five cherry viruses from sweet cherry trees in Japan. Journal of General Plant Pathology, 2004, 70(5): 288-291.

[3]Sabanadzovic S, Abou Ghanem-Sabanadzovic N, Rowhani A, Grant J A, Uyemoto J K. Detection of Chery virus A, Cherry necrotic rusty mottle virus and Little cherry virus 1 in California orchards. Journal of Plant Pathology, 2005, 87(3): 173-177.

[4]Bajet N B, Unruh T R, Druffel K L, Eastwell K C. Occurrence of two little cherry viruses in sweet cherry in Washington State. Plant Disease, 2008, 92(2): 234-238.

[5]Cevik B, Yardimci N, Culal-Kilic H. Detection of viruses infecting stone fruits in Western Mediterranean region of Turkey. Plant Pathology Journal, 2011, 27(1): 44-52.

[6]李明福, 张永江, 黄冲, 相宁, 张秀琴, 李桂芬, 魏梅生, 陈燕芳. 北京怀柔地区樱桃上发现李属坏死环斑病毒. 植物病理学报, 2005, 35(6): 552-554.

Li M F, Zhang Y J, Huang C, Xiang N, Zhang X Q, Li G F, Wei M S, Chen Y F. Prunus necrotic ringspot virus found in cherry in Huairou, Beijing. Acta Phytopathologica Sinica, 2005, 35(6): 552-554. (in Chinese)

[7]代红艳, 张志宏, 吴禄平, 侯义龙, 吕德国. 甜樱桃茎尖培养及PNRSV的RT-PCR检测. 园艺学报, 2003, 30(1): 87-89.

Dai H Y, Zhang Z H, Wu L P, Hou Y L, Lü D G. In vitro shoot tip culture of sweet cherry cultivars and detection of PNRSV by RT-PCR. Acta Horticulturae Sinica, 2003, 30(1): 87-89. (in Chinese)

[8]侯义龙, 张开春, 杨俊玲. 应用RT-PCR方法检测桃和樱桃及其组培苗上的PNRSV和PDV. 果树学报, 2005, 22(3): 292-293.

Hou Y L, Zhang K C, Yang J L. Detection of PNRSV and PDV in peach and cherry by RT-PCR. Journal of Fruit Science, 2005, 22(3): 292-293. (in Chinese)

[9]侯义龙, 杨俊玲, 李春敏. 李矮缩病毒RT-PCR方法建立及检测应用. 中国农业科学, 2005, 38(2): 425-427.

Hou Y L, Yang J L, Li C M. Establishment and application of detection of Prune Dwarf Virus (PDV) by RT-PCR. Scientia Agricultura Sinica, 2005, 38(2): 425-427. (in Chinese)

[10]赵世恒, 王进忠, 李明福, 李桂芬, 马洁, 勾军伟. 北京怀柔地区李矮缩病毒的检测鉴定. 中国植保导刊, 2009, 29(2): 5-7.

Zhao S H, Wang J Z, Li M F, Li G F, Ma J, Gou J W. Detection and identification of Prune dwarf virus in Huairou district in Beijing. China Plant Protection, 2009, 29(2): 5-7. (in Chinese)

[11]Rao W L, Li F, Zuo R J, Li R. First report of Little cherry virus 2 in flowering and sweet cherry trees in China. Plant Disease, 2011, 95(11): 1484.

[12]李青, 覃兰荚, 李明福, 张成良, 李桂芬, 相红. 酶联免疫法检测核果果树病毒. 北京农业科学, 1996, 14(4): 33-35.

Li Q, Qin L J, Li M F, Zhang C L, Li G F, Xiang H. Detection and identification of stone fruit viruses by ELISA. Beijing Agricultural Sciences, 1996, 14(4): 33-35. (in Chinese)

[13]阮小凤, 杨勇, 马书尚, 周瑗月. 甜樱桃病毒病的ELISA检测研究. 山东农业大学学报, 1998, 29(3): 277-282.

Ruan X F, Yang Y, Ma S S, Zhou Y Y. Researches on virus detection by ELISA in sweet cherry. Journal of Shandong Agricultural University, 1998, 29(3): 277-282. (in Chinese)

[14]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[D]. 沈阳: 沈阳农业大学, 2000.

Hou Y L. Development and optimization of detection of fruit tree viruses and cloning of virus-specific DNA fragments[D]. Shenyang: Shenyang Agricultural University, 2000. (in Chinese)

[15]宗晓娟, 王文文, 陈立伟, 王甲威, 严雪瑞, 刘庆忠. 两种主要甜樱桃病毒RT-PCR检测方法的改进. 植物保护学报, 2011, 38(3): 285-286.

Zong X J, Wang W W, Chen L W, Wang J W, Yan X R, Liu Q Z. Improvement of detection method for two main viruses in sweet cherry by RT-PCR. Acta Phytophylacica Sinica, 2011, 38(3): 285-286. (in Chinese)

[16]Noorania M S, Awasthia P, Sharma M P, Rama R, Zaidia A A, Hallana V. Simultaneous detection and identification of four cherry viruses by two step multiplex RT-PCR with an internal control of plant nad5 mRNA. Journal of Virological Methods, 2013, 193(1): 103-107.

[17]Dai J, Cheng J, Huang T, Zheng X, Wu Y. A multiplex reverse transcription PCR assay for simultaneous detection of five tobacco viruses in tobacco plants. Journal of Virological Methods, 2012, 183(1): 57-62.

[18]Li F, Zuo R, Abad J, Xu D, Bao G, Li R. Simultaneous detection and differentiation of four closely related sweet potato potyviruses by a multiplex one-step RT-PCR. Journal of Virological Methods, 2012, 186: 161-166.

[19]Song A, You Y, Chen F, Li P, Jiang J, Chen S. A multiplex RT-PCR for rapid and simultaneous detection of viruses and viroids in chrysanthemum. Letters in Applied Microbiology, 2012, 56(1): 8-13.

[20]Ge B, Li Q, Liu G, Lu M, Li S, Wang H. Simultaneous detection and identification of four viruses infecting pepino by multiplex RT-PCR. Archives of Virology, 2013, 158(6): 1181-1187.

[21]Yu Y, Zhao Z, Jiang D, Wu Z, Li S. A one-step multiplex RT-PCR assay for simultaneous detection of four viruses that infect peach. Letters in Applied Microbiology, 2013, 57(4): 350-355.

[22]中华人民共和国进境植物检疫性有害生物名录. 中华人民共和国农业部公告第862号, 2007-05-29.

Pest lists in import quarantine plants of People’s Republic of China. Ministry of Agriculture No. 862, 2007-05-29. (in Chinese)

[23]董薇, 宋雅坤, 吴明勤, 王林, 吴元华. 大樱桃病毒病研究进展. 中国农学通报, 2005, 21(5): 332-336.

Dong W, Song Y K, Wu M Q, Wang L, Wu Y H. Advances in research of cherry virus diseases. Chinese Agricultural Science Bulletin, 2005, 21(5): 332-336. (in Chinese)

[24]Vasková D, Petrzik K, Karesová R. Variability and molecular typing of the woody-tree infecting Prunus necrotic ringspot ilarvirus. Archives of Virology, 2000, 145(4): 699-709.

[25]Rott M E, Jelkmann W. Detection and partial characterization of a second closterovirus associated with little cherry disease, Little cherry virus-2. Phytopathology, 2001, 91: 261-267.