同步检测<em>PMWaV-1、PMWaV-2</em>和<em>PMWaV-3</em>的三重RT-qPCR方法的初步建立

doi:10.7668/hbnxb.2017.02.006

华北农学报 ›› 2017, Vol. 32 ›› Issue (2): 38-44. doi: 10.7668/hbnxb.2017.02.006

同步检测PMWaV-1、PMWaV-2和PMWaV-3的三重RT-qPCR方法的初步建立

胡加谊^1,2, 罗志文¹, 何凡¹, 李向宏¹, 胡福初¹, 范鸿雁¹, 刘志昕³, 张治礼¹

1. 海南省农业科学院热带果树研究所, 农业部海口热带果树科学观测实验站, 海南省热带果树生物学重点实验室, 海南海口 571100;
2. 黄埔出入境检验检疫局, 广东广州 510730;
3. 中国热带农业科学院热带生物技术研究所, 海南海口 571101

收稿日期:2017-01-20 出版日期:2017-04-28
通讯作者: 张治礼(1970-),男,安徽临泉人,研究员,博士,主要从事果树生理与分子生物学研究。
作者简介:胡加谊(1988-),男,广东梅州人,研究实习员,硕士,主要从事果树植物病理学研究。
基金资助:
海南省重点研发项目（ZDYF2016035）；公益性行业（农业）科研专项经费项目（201203021）；国家自然科学基金项目（31260460）

Establishment of a Triple RT-qPCR Method for the Simultaneous Detecting of PMWaV-1, PMWaV-2 and PMWaV-3

HU Jiayi^1,2, LUO Zhiwen¹, HE Fan¹, LI Xianghong¹, HU Fuchu¹, FAN Hongyan¹, LIU Zhixin³, ZHANG Zhili¹

1. Institute of Tropical Fruit Trees in Hainan Academy of Agricultural Sciences, Haikou Investigation Station of Tropical Fruit Trees in Ministry of Agriculture, Key Laboratory of Tropical Fruit Tree Biology of Hainan Province, Haikou 571100, China;
2. Huangpu Entry-Exit Inspection and Quarantine Bureua, Guangzhou 510730, China;
3. Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China

Received:2017-01-20 Published:2017-04-28

摘要/Abstract

摘要： 目前我国菠萝产区已相继报道3种菠萝凋萎相关病毒（PMWaV-1、PMWaV-2和PMWaV-3）。通过建立这3种菠萝凋萎相关病毒实时荧光定量RT-PCR（RT-qPCR）同步定量检测方法，为菠萝凋萎病毒的定性定量研究提供技术手段。根据这3种菠萝凋萎相关病毒的外壳蛋白基因序列分别设计特异性引物和TaqMan水解探针，在优化PMWaV-1、PMWaV-2、PMWaV-3三重RT-qPCR反应体系后，以PMWaV-1、PMWaV-2、PMWaV-3 CP基因目的片段的重组质粒为标准品绘制标准曲线，初步建立了PMWaV-1、PMWaV-2、PMWaV-3三重RT-qPCR检测方法。其中PMWaV-1标准曲线为y=-3.283logx+39.02，其扩增效率达101.7%，线性相关系数R²=0.999；PMWaV-2标准曲线为y=-3.393 logx+37.53，其扩增效率为97.1%，线性相关系数R²=0.998；PMWaV-3标准曲线为y=-3.171 logx+38.48，其扩增效率为106.7%，线性相关系数R²=0.996；这表明3种菠萝凋萎相关病毒质粒标准品在同一反应体系中可稳定扩增，且线性关系表现良好，可用于后续试验。灵敏度试验数据表明，该方法对PMWaV-1、PMWaV-2、PMWaV-3的最低检测线分别为99，98，868拷贝；重复性试验表明PMWaV-1、PMWaV-2、PMWaV-3三重RT-qPCR扩增曲线的标准差小于0.267，变异系数小于1.44%。这表明建立的三重RT-qPCR检测方法可快速、准确、稳定地定量检测PMWaV-1、PMWaV-2、PMWaV-3等3种病毒，为PMWaV的定性定量研究和复合侵染机制的探索提供了技术基础。

关键词: 菠萝凋萎病, 检测方法, 实时荧光定量RT-PCR, PMWaV, TaqMan探针

Abstract: The objective of this paper was to provide a technique for quantitative and quanlitative studies of PMWaV by establishing the method of Real-time Fluorescent Quantitative RT-PCR (RT-qPCR)for simultaneous quantitative detection of PMWaV-1,PMWaV-2 and PMWaV-3.Primers and probes were designed and synthesized according to the CP gene of PMWaV-1,PMWaV-2 and PMWaV-3,respectively.After optimized the reaction system of triple RT-qPCR,the standard curves were generated by purified DNA of the recombined plasmid of PMWaV-1,PMWaV-2,PMWaV-3 coat protein gene,respectively.And the method of RT-qPCR for the simultaneous detection of PMWaV-1,PMWaV-2,PMWaV-3 had been improved.The amplification efficiency of PMWaV-1,PMWaV-2,PMWaV-3 in the RT-qPCR method was up to 101.7%,97.1%,106.7%,respectively.Meanwhile,with the R² between 0.996 and 0.999.The sensibility test of triple RT-qPCR syetem showed the detection limit of quantification for PMWaV-1,PMWaV-2,PMWaV-3 was 99,98 and 868 copies,respectively.Moreover,repeats experiment datas showed that standard deviation was below 0.26 and variable coefficient was below 1.44%.This study indicated that the method for simultaneous detection and quantification of PMWaV-1,PMWaV-2,PMWaV-3 was a fast,sably and sensitive reaction system,which could be the technological base for quantitative and quanlitative studies of PMWaV.

Key words: Mealybug Wilt of Pineapple, Dctection method, RT-qPCR, PMWaV, TaqMan probe

中图分类号:

S432.4
Q78

胡加谊, 罗志文, 何凡, 李向宏, 胡福初, 范鸿雁, 刘志昕, 张治礼. 同步检测PMWaV-1、PMWaV-2和PMWaV-3的三重RT-qPCR方法的初步建立[J]. 华北农学报, 2017, 32(2): 38-44. doi: 10.7668/hbnxb.2017.02.006.

HU Jiayi, LUO Zhiwen, HE Fan, LI Xianghong, HU Fuchu, FAN Hongyan, LIU Zhixin, ZHANG Zhili. Establishment of a Triple RT-qPCR Method for the Simultaneous Detecting of PMWaV-1, PMWaV-2 and PMWaV-3[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2017, 32(2): 38-44. doi: 10.7668/hbnxb.2017.02.006.

http://www.hbnxb.net/CN/Y2017/V32/I2/38

参考文献

[1] Melzer M,Karasev A,Sether D,et al.Nucleotide sequence,genome organization and phylogenetic analysis of pineapple mealybug wilt-associated virus-2[J].The Journal of General Virology,2001,82(Pt 1):1-7.
[2] Melzer M,Sether D,Karasev A,et al.Complete nucleotide sequence and genome organization of pineapple mealybug wilt-associated virus-1[J].Archives of Virology,2008,153(4):707-714.
[3] 徐迟默.菠萝凋萎病毒研究进展[J].热带作物学报,2009,30(5):718-724.
[4] 李运合,莫忆伟,习金根,等.应用RT-PCR方法检测菠萝凋萎病病毒[J].热带作物学报,2010,31(6):1003-1008.
[5] 吴丽亭,杨晓琪,杨贞爱,等.我国南方三省菠萝病毒种类的分子鉴定[C]//中国植物病理学会2010年学术年会论文集,厦门:中国农业科学技术出版社,2010:455.
[6] Sether D M,Melzer M J,Busto J,et al.Diversity and mealybug transmissibility of ampeloviruses in pineapple[J].Plant Disease,2005,89(5):450-456.
[7] Gambley C F,Steele V,Geering A,et al.The genetic diversity of ampeloviruses in Australian pineapples and their association with mealybug wilt disease[J].Australasian Plant Pathology,2008,37(2):95-105.
[8] Sether D,Hu J.Closterovirus infection and mealybug exposure are necessary for the development of mealybug wilt of pineapple disease[J].Phytopathology,2002,92(9):928-935.
[9] Sipes B S,Sether D M,Hu J S.Interactions between Rotylenchus reniformis and Pineapple mealybug wilt associated virus-1 in pineapple[J].Plant Disease,2002,86(9):933-938.
[10] 胡加谊,罗志文,范鸿雁,等.菠萝凋萎相关病毒-2实时荧光定量RT-PCR检测方法的建立[J].园艺学报,2014,41(6):1257-1266.
[11] 胡加谊,李向宏,罗志文,等.菠萝凋萎相关病毒-3实时荧光定量PCR检测方法的建立[J].果树学报,2015(1):156-162.
[12] 胡加谊,罗志文,李向宏,等.菠萝凋萎相关病毒-1实时荧光定量RT-PCR检测体系的建立与应用[J].植物保护,2014(6):116-121, 125.
[13] 罗志文.海南省菠萝主要病原真菌鉴定及病原病毒多重RT-PCR检测体系的建立[D].海口:海南大学,2011.
[14] 苗立中, 沈志强, 韩文瑜. 副猪嗜血杆菌荧光定量PCR检测方法的建立[J].动物医学进展, 2012, 33(11):85-89.
[15] 伍妙梨, 袁文, 饶丹,等. 胆汁螺杆菌实时荧光定量PCR检测方法的建立[J].中国比较医学杂志, 2015(10):59-63.
[16] Gunasinghe U B,German T L.Purification and partial characterization of a virus from pineapple[J].Phytopathology,1989(70):1337-1341.
[17] Hu J S,Sether D M,Liu X P,et al.Use of a tissue blotting immunoassay to examine the distribution of pineapple closterovirus in Hawaii[J].Plant Disease,1997,81(10):1150-1154.
[18] Hu J S,Sether D M,Ullman D E.Detection of pineapple closterovirus in pineapple plants and mealybugs using monoclonal antibodies[J].Plant Pathology,1996,45(5):829-836.
[19] 郭立新,向本春,朱水芳.苹果茎沟病毒实时荧光RT-PCR反应体系的优化[J].北方园艺,2009(3):8-11.
[20] Henegariu O,Heerema N,Dlouhy S,et al.Multiplex PCR:critical parameters and step-by-step protocol[J].BioTechniques,1997,23(3):504-511.
[21] Johnson K L,Walcott R R.Progress towards a Real-time PCR assay for the simultaneous detection of clavibacter michiganensis subsp michiganensis and pepino Mosaic virus in tomato seed[J].Journal of Phytopathology,2012,160(7/8):353-363.
[22] Arezi B,Xing W,Sorge J,et al.Amplification efficiency of thermostable DNA polymerases[J].Analytical Biochemistry,2003,321(2):226-235.

选择文件类型/文献管理软件名称

选择包含的内容

同步检测PMWaV-1、PMWaV-2和PMWaV-3的三重RT-qPCR方法的初步建立

Establishment of a Triple RT-qPCR Method for the Simultaneous Detecting of PMWaV-1, PMWaV-2 and PMWaV-3

PDF

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 2

编辑推荐

Metrics

本文评价

[1]	梅鸿献, 刘艳阳, 武轲, 杜振伟, 郑永战. 芝麻种子老化处理生理特性变化及种子活力适宜检测方法研究[J]. 华北农学报, 2013, 28(5): 169-174.
[2]	陈金松, 黄丛林, 张秀海, 吴忠义. 环介导等温扩增技术检测含有CaMV35S的转基因玉米[J]. 华北农学报, 2011, 26(4): 8-14.

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

同步检测PMWaV-1、PMWaV-2和PMWaV-3的三重RT-qPCR方法的初步建立

Establishment of a Triple RT-qPCR Method for the Simultaneous Detecting of PMWaV-1, PMWaV-2 and PMWaV-3

PDF

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 2

编辑推荐

Metrics

本文评价