甘薯褪绿矮化病毒西非株系RT-LAMP检测方法的建立

doi:10.3864/j.issn.0578-1752.2013.18.025

中国农业科学 ›› 2013, Vol. 46 ›› Issue (18): 3939-3945.doi: 10.3864/j.issn.0578-1752.2013.18.025

甘薯褪绿矮化病毒西非株系RT-LAMP检测方法的建立

乔奇, 张振臣, 秦艳红, 张德胜, 田雨婷, 王爽, 王永江

河南省农业科学院植物保护研究所/河南省农作物病虫害防治重点实验室/农业部华北南部作物有害生物综合治理重点实验室，郑州 450002

收稿日期:2013-03-26 出版日期:2013-09-15 发布日期:2013-06-17
通讯作者: 通信作者张振臣，Tel：0371-65711547；E-mail：zhangzhenchen@126.com
作者简介:乔奇，E-mail：qiaoq2005@126.com
基金资助:
国家甘薯产业技术体系建设项目（CARS-11-B-07）

Detection of Sweet potato chlorotic stunt virus Strain WA by Reverse Transcription Loop-Mediated Isothermal Ampli?cation Reaction

QIAO Qi, ZHANG Zhen-Chen, QIN Yan-Hong, ZHANG De-Sheng, TIAN Yu-Ting, WANG Shuang, WANG Yong-Jiang

Institute of Plant Protection, Henan Academy of Agricultural Sciences/Henan Key Laboratory of Crop Pest Control/IPM Key Laboratory in Southern Part of North China for Ministry of Agriculture, Zhengzhou 450002

Received:2013-03-26 Online:2013-09-15 Published:2013-06-17

摘要/Abstract

摘要： 【目的】基于逆转录环介导等温扩增技术（Reverse transcription loop-mediated isothermal amplification，RT-LAMP）建立甘薯褪绿矮化病毒（Sweet potato chlorotic stunt virus，SPCSV）西非株系（West African，WA）的可视化检测方法。【方法】针对SPCSV西非株系（SPCSV-WA）的CP核苷酸序列设计4条引物，以感染SPCSV-WA甘薯叶片总RNA为模板，进行一步法RT-LAMP，在65℃条件下反应1 h。扩增产物利用琼脂糖电泳分析和SYBR green I荧光染料显色判断结果，同时对扩增产物的电泳条带进行基因克隆和测序鉴定。利用常规RT-PCR和建立的RT-LAMP方法对14份甘薯样品进行SPCSV-WA检测，比较2种方法的检测结果。【结果】建立的LAMP方法可特异地对SPCSV-WA进行检测，且最低可检测出101拷贝/µL的模板。RT-LAMP产物的克隆测序表明，感染SPCSV-WA甘薯样品的RT-LAMP产物为SPCSV-WA CP基因序列。14份田间甘薯样品检测表明，RT-LAMP与RT-PCR方法检测结果完全一致。【结论】建立的SPCSV-WA RT-LAMP可视化检测方法，是简便、可靠的SPCSV-WA检测方法。

关键词: 甘薯 , 甘薯褪绿矮化病毒西非株系 , 逆转录环介导等温扩增技术 , 检测

Abstract: 【Objective】The objective of this study is to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of Sweet potato chlorotic stunt virus strain WA (SPCSV-WA). 【Method】Four RT-LAMP primers, designed on the basis of the coat protein (CP) gene sequences of SPCSV-WA, and template RNA from infected leaves were used for one-step RT-LAMP which were carried out under isothermal conditions at 65℃ for 1 h. RT-LAMP products were analyzed by electrophoresis in agarose gels followed by staining with SYBR green I. A fragment from the RT-LAMP product was then cloned and sequenced to con?rm the spect?city of the assay. The RT-LAMP, and meanwhile compared with the conventional RT-PCR assay, was performed to detect SPCSV-WA from 14 sweet potato samples. 【Result】The RT-LAMP showed a high specificity and the detection limit was 101 copies/µL RNA template. A fragment cloned from the RT-LAMP product indicated that the nucleotide sequence cloned was SPCSV-WA CP. Fourteen sweet potato samples testing results of RT-LAMP were consistent with that of RT-PCR. 【Conclusion】 The RT-LAMP described in this study represents a very sensitive, speci?c and rapid assay for the detection of SPCSV-WA.

Key words: Ipomoea batatas , Sweet potato chlorotic stunt virus strain WA , RT-LAMP , detection

乔奇, 张振臣, 秦艳红, 张德胜, 田雨婷, 王爽, 王永江. 甘薯褪绿矮化病毒西非株系RT-LAMP检测方法的建立[J]. 中国农业科学, 2013, 46(18): 3939-3945.

QIAO Qi, ZHANG Zhen-Chen, QIN Yan-Hong, ZHANG De-Sheng, TIAN Yu-Ting, WANG Shuang, WANG Yong-Jiang. Detection of Sweet potato chlorotic stunt virus Strain WA by Reverse Transcription Loop-Mediated Isothermal Ampli?cation Reaction[J]. Scientia Agricultura Sinica, 2013, 46(18): 3939-3945.

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.0578-1752.2013.18.025

https://www.chinaagrisci.com/CN/Y2013/V46/I18/3939

参考文献

[1]Clark C A, Davis J A, Abad J A, Cuellar W J, Fuentes S, Kreuze J F, Gibson R W, Mukasa S B, Tugume A K, Tairo F D, Valkonen J P T. Sweetpotato viruses: 15 years of progress on understanding and managing complex diseases. Plant Disease, 2012, 96(2): 168-185.

[2]Mukasa S B, Rubaihayo P R, Valkonen J P T. Interactions between a crinivirus, an ipomovirus and a potyvirus in coinfected sweetpoato plants. Plant Pathology, 2006, 55: 458-467.

[3]Untiveros M, Fuentes S, Salazar L F. Synergistic interaction of Sweet potato chlorotic stunt virus (Crinivirus) with carla-, cucumo-, ipomo-, and potyviruses infecting sweet potato. Plant Disease, 2007, 91(6): 669-676.

[4]Gutiérrez D L, Fuentes S, Salazar L F. Sweetpotato virus disease (SPVD): Distribution, incidence, and effect on sweetpotato yield in Peru. Plant Disease, 2003, 87(3): 297-302.

[5]Njeru R W, Mburu M W K, Cheramgoi E, Gibson R W, Kiburi Z M, Obudho E, Yobera D. Studies on the physiological effects of viruses on sweet potato yield in Kenya. Annals of Applied Biology, 2004, 145: 71-76.

[6]张振臣, 乔奇, 秦艳红, 张德胜, 田雨婷. 我国发现由甘薯褪绿矮化病毒和甘薯羽状斑驳病毒协生共侵染引起的甘薯病毒病害. 植物病理学报, 2012, 42(3): 328-333.

Zhang Z C, Qiao Q, Qin Y H, Zhang D S, Tian Y T. First evidence for occurrence of Sweet potato virus disease (SPVD) caused by dual infection of Sweet potato feathery mottle virus and Sweet potato chlorotic stunt virus in China. Acta Phytopathologica Sinica, 2012, 42(3): 328-333. (in Chinese)

[7]Qin Y H, Zhang Z C, Qiao Q, Zhang D S, Tian Y T, Wang Y J. Molecular variability of Sweet potato chlorotic stunt viruses (SPCSV) and five potyviruses infecting sweet potato in China. Archives of Virology, 2013, 158(2): 491-495.

[8]Kokkinos C D, Clark C A. Real-time PCR assays for detection and quantification of sweetpotato viruses. Plant Disease, 2006, 90(6): 783-788.

[9]Mukasa S B, Rubaihayo P R. Valkonen J P T. Incidence of viruses and viruslike diseases of sweetpotato in Uganda. Plant Disease, 2003, 87(4): 329-335.

[10]Aritua V, Bua B, Barg E, Vetten H J, Adipala E, Gibson R W. Incidence of ?ve viruses infecting sweetpotatoes in Uganda; the ?rst evidence of Sweet potato caulimo-like virus in Africa. Plant Pathology, 2007, 56: 324 -331.

[11]Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanebe K, Amino N, Hase T. Loop-mediated isothermal ampli?cation of DNA. Nucleic Acids Research, 2000, 28(12): e63.

[12]Fukuta S, Iida T, Mizukami Y, Ishida A, Ueda J, Kanbe M, Ishimoto Y. Detection of Japanese yam mosaic virus by RT-LAMP. Archives of Virology, 2003, 148(9): 1713-1720.

[13]Zhao L, Cheng J, Hao X, Tian X, Wu Y. Rapid detection of tobacco viruses by reverse transcription loop-mediated isothermal amplification. Archives of Virology, 2012, 157(12): 2291-2298.

[14]Peng J, Shi M, Xia Z, Huang J, Fan Z. Detection of cucumber mosaic virus isolates from banana by one-step reverse transcription loop-mediated isothermal amplification. Archives of Virology, 2012, 157(11): 2213-2217.

[15]周彤, 杜琳琳, 范永坚, 周益军. 水稻黑条矮缩病毒RT-LAMP快速检测方法的建立. 中国农业科学, 2012, 45(7): 1285-1292.

Zhou T, Du L L, Fan Y J, Zhou Y J. Development of a RT-LAMP assay for rapid detection of Rice black-streaked dwarf virus. Scientia Agricultura Sinica, 2012, 45(7): 1285-1292. (in Chinese)

[16]Yang L, Li J, Bi Y, Xu L, Liu W. Development and application of a reverse transcription loop-mediated isothermal ampli?cation method for rapid detection of Duck hepatitis A virus type 1. Virus Genes, 2012, 45(3): 585-589.

[17]Komiyama C, Suzuki K, Miura Y, Sentsui H. Development of loop-mediated isothermal amplification method for diagnosis of bovine leukemia virus infection. Journal of Virological Methods, 2009, 157(2): 175-179.

[18]Rovira A, Abrahante J, Murtaugh M, Munoz-Zanzi C. Reverse transcription loop-mediated isothermal ampli?cation for the detection of Porcine reproductive and respiratory syndrome virus. Journal of Veterinary Diagnostic Investigation, 2009, 21: 350-354.

[19]Hoyer U, Maiss E, Jelkmann W, Lesemann D E, Vetten H J. Identification of the coat protein gene of a sweet potato sunken vein closterovirus isolate from Kenya and evidence for a serological relationship among geographically diverse closterovirus isolates from sweet potato. Phytopathology, 1996, 86(7): 744-750.

[20]Aritua V, Barg E, Adipala E, Gibson R W, Vetten H J. Further evidence for limited genetic diversity among East African isolates of Sweet potato chlorotic stunt virus. Journal of Phytopathology, 2008, 156: 181-189.

[21]Tairo F, Mukasa S B, Jones R A C, Kullaya A, Rubaihayo P R, Valkonen J P T. Unravelling the genetic diversity of the three main viruses involved in sweet potato virus disease (SPVD), and its practical implications. Molecular Plant Pathology, 2005, 6(2): 199-211.

[22]Qiao Q, Zhang Z C, Qin Y H, Zhang D S, Tian Y T, Wang Y J. First report of Sweet potato chlorotic stunt virus infecting sweet potato in China. Plant Disease, 2011, 95(3): 356.

[23]Hong T C, Mai Q L, Cuong D V, Parida M, Minekawa H, Notomi T, Hasebe F, Morita K. Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus. Journal of Clinical Microbiology, 2004, 42(5): 1956-1961.

甘薯褪绿矮化病毒西非株系RT-LAMP检测方法的建立

Detection of Sweet potato chlorotic stunt virus Strain WA by Reverse Transcription Loop-Mediated Isothermal Ampli?cation Reaction

PDF

赞

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

Metrics

本文评价

推荐阅读 0

[1]	王一丹,杨发龙,陈弟诗,向华,任玉鹏. 猪腹泻病毒一步法多重TaqMan荧光定量RT-PCR检测法的建立及应用[J]. 中国农业科学, 2023, 56(1): 179-192.
[2]	李志玲,李香菊,崔海兰,于海燕,陈景超. 牛筋草EPSPS酶联免疫试剂盒的研发及应用[J]. 中国农业科学, 2022, 55(24): 4851-4862.
[3]	谢丽雪,张小艳,张立杰,郑姗,李韬. 侵染西番莲的东亚西番莲病毒全基因组序列特征及TC-RT-PCR检测技术[J]. 中国农业科学, 2022, 55(22): 4408-4418.
[4]	崔鹏,赵逸人,姚志鹏,庞林江,陆国权. 低温对甘薯淀粉理化特性及代谢关键基因表达量的影响[J]. 中国农业科学, 2022, 55(19): 3831-3840.
[5]	崔江宽,任豪豪,曹梦园,陈昆圆,周博,蒋士君,汤继华. 玉米孢囊线虫SCAR-PCR快速分子检测技术[J]. 中国农业科学, 2022, 55(17): 3334-3342.
[6]	范文静,刘明,赵鹏,张强强,吴德祥,郭鹏宇,朱晓亚,靳容,张爱君,唐忠厚. 甘薯苗期耐低氮基因型筛选及不同氮效率类型综合评价[J]. 中国农业科学, 2022, 55(10): 1891-1902.
[7]	张静远,缪发明,陈腾,李敏,扈荣良. 非洲猪瘟实时荧光RPA诊断方法建立及应用[J]. 中国农业科学, 2022, 55(1): 197-207.
[8]	李镇希,李文婷,黄家权,郑正,许美容,邓晓玲. 膜吸附法结合可视化环介导等温扩增技术检测柑橘黄龙病菌[J]. 中国农业科学, 2022, 55(1): 74-84.
[9]	段玉,许建建,马志敏,宾羽,周常勇,宋震. 柑橘叶斑驳病毒的逆转录重组酶聚合酶扩增检测[J]. 中国农业科学, 2021, 54(9): 1904-1912.
[10]	白雪,惠腾,王振宇,曹云刚,张德权. 高效液相色谱-荧光检测法检测烤肉制品中5种硝基多环芳烃[J]. 中国农业科学, 2021, 54(5): 1055-1062.
[11]	李佳佳,洪慧龙,万明月,储丽,赵敬会,汪明华,徐志鹏,张阴,黄志平,张文明,王晓波,邱丽娟. 基于近红外光谱的大豆茎秆化学组分含量检测模型构建与应用[J]. 中国农业科学, 2021, 54(5): 887-900.
[12]	王欣,李强,曹清河,马代夫. 中国甘薯产业和种业发展现状与未来展望[J]. 中国农业科学, 2021, 54(3): 483-492.
[13]	赵珊,仲伶俐,秦琳,黄世群,李曦,郑幸果,雷欣宇,雷绍荣,郭灵安,冯俊彦. 不同干燥方式对甘薯叶功能成分及抗氧化活性的影响[J]. 中国农业科学, 2021, 54(21): 4650-4663.
[14]	靳容,刘明,赵鹏,张强强,张爱君,唐忠厚. 甘薯丝裂原活化蛋白激酶MPK6对低温胁迫的响应[J]. 中国农业科学, 2021, 54(20): 4265-4273.
[15]	赵付枚,王爽,田雨婷,乔奇,王永江,张德胜,张振臣. 甘薯病毒病发生关键因素研究[J]. 中国农业科学, 2021, 54(15): 3232-3240.