逆转录环介导等温扩增技术检测齿兰环斑病毒

樊荣辉; 罗远华; 钟淮钦; 叶秀仙; 黄敏玲

doi:10.19303/j.issn.1008-0384.2019.07.011

逆转录环介导等温扩增技术检测齿兰环斑病毒

doi: 10.19303/j.issn.1008-0384.2019.07.011

樊荣辉^{1, 2, 3,},
罗远华^{1, 2, 3},
钟淮钦^{1, 2, 3},
叶秀仙^{1, 2, 3},
黄敏玲^{1, 2, 3, ,}

1.
福建省农业科学院作物研究所, 福建福州 350013
2.
福建省农业科学院花卉研究中心, 福建福州 350013
3.
福建省特色花卉工程技术研究中心, 福建福州 350013

基金项目:

福建省科技计划项目——省属公益类科研院所基本科研专项 2017R1026-8

福建省财政专项——福建省农业科学院科技创新团队建设项目 STIT2017-2-9

福建省农业科学院生产性工程化实验室中试基金 AG2017-2

详细信息

作者简介:
樊荣辉(1983-), 女, 硕士, 助理研究员, 研究方向:花卉生物技术研究(E-mail:rhfan1012@163.com)

通讯作者:
黄敏玲(1960-), 女, 研究员, 研究方向:花卉种质创新、资源评价及生物技术研究(E-mail:huangml618@163.com)

中图分类号: S436
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- 被引次数: 0
出版历程
- 收稿日期: 2018-04-11
- 修回日期: 2019-03-19
- 刊出日期: 2019-07-20

RT-LAMP Detection of Odontoglossum Ringspot Virus

FAN Rong-hui^{1, 2, 3
,},
LUO Yuan-hua^{1, 2, 3},
ZHONG Huai-qin^{1, 2, 3},
YE Xiu-xian^{1, 2, 3},
HUANG Min-ling^{1, 2, 3
, ,}

1.
Institute of Crop Sciences, Fujian Academy of Agricultural Science, Fuzhou, Fujian 350013, China
2.
Flowers Research Center, Fujian Academy of Agricultural Science, Fuzhou, Fujian 350013, China
3.
Fujian Engineering Research Center for Characteristic Floriculture, Fuzhou, Fujian 350013, China

摘要

摘要: 目的齿兰环斑病毒Odontoglossum ringspot virus（ORSV）是侵染兰花的主要病毒，严重影响其观赏价值，建立快速、灵敏的检测方法显得尤为重要。方法本研究根据ORSV的外壳蛋白基因序列设计了一组特异性引物，经过一系列条件优化，建立了该病毒的RT-LAMP（Reverse Transcription-Loop-mediated Iisothermal Amplification）检测方法。结果结果显示该方法能特异扩增ORSV，与其他4种侵染兰花的病毒（建兰花叶病毒、菜豆黄花叶病毒、黄瓜花叶病毒和小苍兰花叶病毒）不发生反应；灵敏度为RT-PCR的10倍。田间检测20份样品中，RT-LAMP和RT-PCR检测结果一致。在产物中加入荧光染料SYBR GreenⅠ直接用肉眼观察即可判断样品是否感染ORSV，省去电泳分析的时间，并且增加了在田间的应用价值。结论该方法具有特异性强、灵敏度高、操作简单、快速等特点。
- 齿兰环斑病毒 /
- RT-LAMP /
- 检测
Abstract: Objective To establish a rapid and reliable detection method on Odontoglossum ringspot virus (ORSV), a major pathogen that seriously affects the ornamental value of orchids. Method Primers were designed for the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay from the conserved region in the coat protein (CP) gene of ORSV available in GenBank, and its reaction conditions optimized. Result Using the selected primer, RT-LAMP specifically identified ORSV but not Cymbidium mosaic virus (CyMV), Bean yellow mosaic virus (BYMV), Freesia mosaic virus (FreMV) or Cucumber mosaic virus (CMV). The sensitivity of the assay was 10 times higher than that of RT-PCR, while identical in positive rate on test of 20 specimens. Furthermore, the amplification products of the newly developed method could be visually inspected using SYBR Green I without gel electrophoresis. Conclusion The RT-LAMP assay was considered a specific, sensitive, and rapid method for ORSV detection.
- Odontoglossum ringspot virus /
- RT-LAMP /
- detection

HTML全文

图 1 RT-LAMP检测ORSV

注：M：DL2000 DNA marker; 1：阴性对照；2：LAMP产物(ORSV)。

Figure 1. Detection of ORSV by RT-LAMP

Note:M:DL2000 DNA marker; 1:Negative control; 2:LAMP product.

下载: 全尺寸图片幻灯片

图 2 LAMP产物酶切鉴定

注：M：DL2000 DNA marker; 1：酶切产物; 2：阴性对照；3：LAMP产物(ORSV)。

Figure 2. Digestion of LAMP products

Note:M:DL2000 DNA marker; 1:LAMP products for EcoRⅠfor 2 h; 2:Negative control; 3:LAMP product.

下载: 全尺寸图片幻灯片

图 3 RT-LAMP特异性分析

注：M：DL2000 DNA marker; 1~5分别为CyMV、BYMV、CMV、FreMV和ORSV。

Figure 3. Specificity of RT-LAMP assay

Note:M:DL2000 DNA marker; 1-5:CyMV、BYMV、CMV、FreMVand ORSV, respectively.

下载: 全尺寸图片幻灯片

图 4 RT-LAMP与RT-PCR灵敏度比较

Figure 4. Sensitivity of RT-LAMP and RT-PCR assays

下载: 全尺寸图片幻灯片

表 1 RT-LAMP检测所用的引物

Table 1. Primers for RT-LAMP assay

引物名称 Primer	引物序列(5′-3′) Sequence	退火温度 Annealing temperature/℃
F3	TGTCTTACACTATTACAGACCCG	57.2
B3	TCATAGCGATAAACTCTGAAGT	54.2
FIP	GGAACTGATTACCCAGAGAATTGGTA-gaattc-TTATTTAAGCTCGGCTTGGGCTG	75.9
BIP	CTGTTCAACAGCAGTTTGCTGATG-gaattc-GCAGGGAACCTACTGGTCAAAG	78.1

下载: 导出CSV

表 2 田间建兰和文心兰样品的检测

Table 2. Field detection on Cymbidium and Oncidium

方法 Method	建兰Cymbidium		检出率 Positive rate/%	文心兰Oncidium		检出率 Positive rate/%
方法 Method	检测样品数 Sample	阳性样品数 Positive sample	检出率 Positive rate/%	检测样品数 Sample	阳性样品数 Positive sample	检出率 Positive rate/%
RT-LAMP	10	4	40.0	10	3	20.0
RT-PCR	10	4	40.0	10	3	20.0

下载: 导出CSV

参考文献(15)

[1]	HU J S, FERREIRA D, WANG M, et al. Detection of cymbidium mosaic virus, odontoglossum ringspot virus, tomato spotted wilt virus, and potyviruses infecting orchids in Hawai[J]. Plant Disease, 1993, 77:464-468. doi: 10.1094/PD-77-0464
[2]	WONG S M, CHENG C G, LEE Y H, et al. Incidence of cymbidium mosaic and odontoglossum ringspot viruses and their significance in orchid cultivation in Singapore[J]. Crop Protection, 1994, 13:235-239. doi: 10.1016/0261-2194(94)90084-1
[3]	ZETTLER F W, KO N J, WISLER G C, et al. Viruses of orchids and their control[J]. Plant Disease, 1990, 74(9):621-626. doi: 10.1094/PD-74-0621
[4]	MENZEL W, JELKMANN W, MAISS E. Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control[J]. Journal of Virological Methods, 2002, 99:81-92. doi: 10.1016/S0166-0934(01)00381-0
[5]	THOMPSON J R, WETZEL S, KLERKS M M, et al. Multiplex RT-PCR detection of four aphid-borne strawberry viruses in Fragaria spp. in combination with a plant mRNA specific internal control[J]. Journal of Virological Methods, 2003, 111(2):85-93. doi: 10.1016/S0166-0934(03)00164-2
[6]	梁敏国, 刘光华.广东兰花病毒病调查和病原检测[J].江西植保, 2004, 27(3):97-100. doi: 10.3969/j.issn.2095-3704.2004.03.001 LIANG M G, LIU G H. Research and Pathogeny Identification on Virus Disease of Orchid in Guangdong Province[J]. Jiangxi Plant Protection, 27(3):97-100.(in Chinese) doi: 10.3969/j.issn.2095-3704.2004.03.001
[7]	柳爱春, 赵芸, 刘超, 等.双重一步法RT-PCR检测2种主要兰花病毒[J].安徽农业科学, 2009, 37(27):12960-12961, 13009. http://d.old.wanfangdata.com.cn/Periodical/ahnykx200927038 LIU A C, ZHAO Y, LIU C, et al. Detection of Two Main Orchid Viruses with the Method of Double One-step RT-PCR[J]. Journal of Anhui Agricultural Sciences, 2009, 37(27):12960-12961, 13009.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/ahnykx200927038
[8]	NOTOMI T, OKAYMA H, MASUBUCHI H. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 2000, 28(12):63. doi: 10.1093/nar/28.12.e63
[9]	HARPER S J, WARD L I, CLOVER G R. Development of LAMP and real-time PCR methods for the rapid detection of Xylella fastidiosa for quarantine and field applications[J]. Phytopathology, 2010, 100(12):1282-1288. doi: 10.1094/PHYTO-06-10-0168
[10]	RANJAN R, KANJAYAN M, SUBRAMANIAM S, et al. Development and evaluation of a one-step reverse transcription-loop mediated isothemal amplication assay(RT-LAMP) for rapid detection of foot and mouth disease virus in India[J]. Virus Disease, 2014, 25(3):358-364. doi: 10.1007/s13337-014-0211-2
[11]	SINGH P, MIRDHA B R, AHUJA V, et al. Loop-mediated isothermal amplification(LAMP) assay for rapid detection of Entamoeba histolytica in amoebic liver abscess[J]. World Microbiol Biotechnol, 2013, 29:27-32. doi: 10.1007/s11274-012-1154-7
[12]	高宏伟, 徐彪, 朱来华, 等.应用LAMP检测方法检测肉制品中的单增李斯特菌[J].食品安全质量检测技术, 2010, 27(1):12-17. http://d.old.wanfangdata.com.cn/Periodical/spaqzljcjs201001003 GAO H W, XU B, ZHU L H, et al. Detection of Listeria monocytogenes in meat by loop-mediated isothermal amplification (LAMP)[J]. Journal of Food Safety and Quality, 2010, 27(1):12-17.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/spaqzljcjs201001003
[13]	ZHANG Y Q, SHAN X X, SHI L, et al. Development of a fim Y-based loop-mediated isothermal amplification assay for detection of Salmonella in food[J]. Food Research international, 2012, 45(2):1011-1015. doi: 10.1016/j.foodres.2011.02.015
[14]	王永, 兰青阔, 赵新, 等.转基因作物外源转基因成分环介导等温扩增技术检测方法的建立及应用[J].中国农业科学, 2009, 42(4):1473-1477. doi: 10.3864/j.issn.0578-1752.2009.04.044 WANG Y, LAN Q K, ZHAO X, et al. Development and Application of Loop-Mediated Isothermal Amplification for Detection of Genetically Modified Crops[J]. Scientia Agricultura Sinica, 2009, 42(4):1473-1477.(in Chinese) doi: 10.3864/j.issn.0578-1752.2009.04.044
[15]	CHEN L, GUO J, WANG Q, et al. Development of the visual loop-mediated isothermal amplification assay for seven genecitally modified maize events and their application in practical samples analysis[J]. Journal of Agricultural and Food Chemistry, 2011, 59(11):5914-5918. doi: 10.1021/jf200459s