ZHAN Yong,YU De-yue.Tissue Blotting and RT-PCR Detection of Soybean Mosaic Virus[J].Soybean Science,2010,29(06):915-919.[doi:10.11861/j.issn.1000-9841.2010.06.0915]
大豆花叶病毒的组织印迹与RT-PCR检测
- Title:
- Tissue Blotting and RT-PCR Detection of Soybean Mosaic Virus
- 文章编号:
- 1000-9841（2010）06-0915-05
- Keywords:
- Soybean mosaic virus; Tissue blotting; RT-PCR; Detection
- 分类号:
- S565.1
- 文献标志码:
- A
- 摘要:
- 用改进的组织印迹方法批量检测黄淮地区69份SMV样品，结果显示42个标样呈现非常清晰的蓝紫色阳性反应，26个标样显色较弱而无法确证，1个标样呈阴性反应。进一步对图片进行600 dpi扫描并放大200倍进行分析，发现26个显色弱的标样中有25个呈阳性反应，1个标样显色很弱仍无法确证，使得检出率提高了37%。利用RT-PCR程序对随机选取的8个样品进行验证，发现纯化与未纯化的RNA模板均能得到较为清晰的扩增条带，而只有纯化后的PCR产物二次扩增才有预期的条带，且双酶切可产生245 bp、255 bp和307 bp 3个预期的片段。表明改进的组织印迹方法能够用于批量、高效地检测SMV，而RT-PCR可用于更为精确的检验。
- Abstract:
- With improved tissue blotting approach, 69 SMV samples from the Yellow and Huaihe River areas were detected and the results showed that 42 samples presented a very clear blue-violet-positive reaction, 26 samples could not made conclusion for weaker color and one sample was negative. Further analysis by 600 dpi scan and enlarging the picture for 200 times, it was observed that 25 from 26 samples suffering from weak color showed positive results, and the negative standard sample were still unable to confirm the color, which makes the detection rate increased by 37%. RT-PCR verification were used in random selected eight samples and the results showed that both purified and unpurified RNA templates could obtain distinct bands, however, only the purified PCR products could be amplified repeatedly with the expected secondary bands, and the double-digestion could also produce three expected fragments, 245 bp, 255 bp and 307 bp. The results above indicated that the improved tissue blotting method could detect soybean SMV efficiently and bulkily, RT-PCR detection could be used in more precise testing.
参考文献/References:
[1]吴云锋. 植物病毒学原理与方法[M]. 西安：西安地图出版社, 1999. (Wu Y F. Principles and methods of plant virology [M]. Xi′an: Xi′an Map Press, 1999.) [2]张仲凯，李毅. 云南植物病毒[M]. 北京：科学出版社, 2001. (Zhang Z K, Li Y. Plant virus in Yunnan [M]. Beijing: Science Press, 2001.) [3] 徐明全，郑平.植物病毒检测技术——组织印迹法[J]. 微生物学通报, 2000, 27(5):360-363.（Xu M Q, Zheng P. A technique for detection of plant viruses-tissue blotting[J]. Microbiology, 2000, 27(5)：360-363.） [4]Hsu H T, Lawson R H. Direct tissue blotting for detection of tomato spotted wilt virus in impatiens [J]. Plant Disease, 1991,75(3):292-295. [5]Lin N S, Hsu Y H, Hsu H T. Immunological detection of plant viruses and a mycoplasmalike organism by direct tissue blotting on nituocellulose membranes [J]. Phytopathological, 1990, 80(9): 824-828. [6]周雪平, 濮祖芹, 方仲达, 等. 大豆花叶病毒外壳蛋白基因克隆及在病毒检测中的应用[J]. 病毒学报，1994,10(1): 81-85. （Zhou X P, Pu Z Q, Fang Z D, et al. Construction and use of cloned soybean mosaic virus(SMV) coat protein gene as probe for the detection of SMV[J]. Chinese Journal of Virology，1994，10(1): 81-85.） [7]赵玖华，尚佑芬，杨崇良, 等. RT-PCR技术检测大豆花叶病毒的研究[J]. 山东农业科学，2000(4): 34-35. （Zhao J H, Shang Y F, Yang C L, et al. Study on the detection of soybean mosaic virus with RT-PCR technique[J]. Shandong Agricultural Sciences, 2000(4): 34-35.） [8]张景凤, 赵慧，桂晋刚，等. 利用RT-PCR、DNA序列分析及原核基因表达对我国大豆花叶病毒进行分子鉴定[J]. 植物学报, 1999, 41(9): 932-935. (Zhang J F, Zhao H, Gui J G, et al. Molecular characterization of a chinese soybean mosaic virus isolate by RT-PCR, cDNA sequence analysis and direct expression of PCR products in bacteria[J]. Acta Botanica Sinica，1999, 41（9）：932-935.) [9]战勇，智海剑，喻德跃，等. 黄淮地区大豆花叶病毒株系的鉴定与分布[J]. 中国农业科学, 2006，39(10)：2009-2015. (Zhan Y，Zhi H J，Yu D Y，et al. Identification and distribution of SMV strains in Huang-Huai valleys[J]. Scientia Agricultura Sinica，2006，39(10)：2009-2015.) [10]杨雅麟. 长江中下游地区大豆花叶病毒(SMV)株系组成、分布及抗性研究[D].南京：南京农业大学，2000. (Yang Y L. Classification and distribution of strains of soybean mosaic virus in the middle and lower Changjiang river valleys and the resistance to soybean mosaic virus in soybeans [D]. Nanjing: Nanjing Agricultural University，2000.) [11]张七仙，刘俊起，李成霞, 等. 一种简捷的同时提取植物总DNA和总RNA的方法[J]. 中国农业科学，2001, 34 (4):458-460. (Zhang Q X, Liu J Q, Li C X, et al. A simple and rapid method for preparing genomic DNA and total RNA from plant tissues[J]. Scientia Agricultura Sinica，2001, 34(4): 458-460.) [12]梁国栋. 最新分子生物学实验技术[M].北京：科学出版社，2001. (Liang G D. Newest molecular biology experimental technique[M]. Beijing: Science Press, 2001.) [13]杨建雄. 生物化学与分子生物学实验技术教程[M].北京：科学出版社, 2002. (Yang J X. Laboratory techniques course of biochemistry and molecular biology[M]. Beijing: Science Press, 2002.) [14]卢圣栋. 现代分子生物学实验技术[M].北京： 高等教育出版社, 1993. ( Lu S D. Current protocols for molecular biology[M]. Beijing: Higher Education Press, 1993.) [15]储瑞银, 冷晓红, 鲍一明, 等. 应用聚合酶链式反应扩增大豆花叶病毒外壳蛋白基因及其序列分析[J]. 植物学报，1992(7):523-527. (Chu R Y, Leng X H, Bao Y M, et al. Amplification of soybean mosaic virus coat protein gene by polymerase chain reaction and its sequence analysis[J]. Acta Botanic Sinica, 1992(7):523-527.) [16]刘俊君，彭学贤，李荔，等. 大豆花叶病毒外壳蛋白基因的克隆和其在大肠杆菌中的表达[J].生物工程学报，1993, 9(3): 198-203.（Liu J J, Peng X X, Li L, et al. Cloning and expression of coat protein gene of soybean mosaic virus in Escherichia coli[J]. Chinese Journal of Biotechnology, 1993, 9(3): 198-203.）
备注/Memo
基金项目：国家重点基础研究发展计划资助项目（2010CB125906）。