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Method for detecting lily seed virus

A detection method, lily technology, applied in the detection of LSV and LMoV, lily ball virus CMV field, can solve the problems of RNA take away, low quality of bulb total RNA extraction, RNA yield reduction, etc., to achieve easy distinction, total Inexpensive and reproducible results

Inactive Publication Date: 2012-07-18
BEIJING FORESTRY UNIVERSITY
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Problems solved by technology

[0002] Application of Multiplex RT-PCR Technology (RT-MPCR) in Lily: Wang Jihua et al. ) detected LSV and LMoV viruses using double PCR technology, Li Haoyan et al. , 32(6), 2006, 42~45) used multiplex RT-PCR to detect LXV, LSV and LMoV, but neither of them detected CMV, and CMV virus is one of the three major viruses infecting lily, which is seriously harmful , widespread disease, pending detection
[0003] Xu Rongxue (Xu Rongxue. Molecular detection and detoxification technology research on main viruses of lily. Nanjing Forestry University, 2007) detected the viruses in the leaves and flowers of cultivated varieties and wild species. The leaves, flowers and scales were extracted with a kit. The extraction quality of total RNA from bulbs is not high, and no RNA extraction method has been established for lily bulbs, and lily bulb tissues are rich in secondary metabolites such as polysaccharides and polyphenols. When the polysaccharides are removed, a gel-like precipitate rich in polysaccharides is often produced. This RNA precipitate containing polysaccharides is difficult to dissolve in water, or a viscous solution will be produced after dissolution. When the polysaccharides are removed, the RNA is also easily taken away, resulting in the loss of RNA. Therefore, it is quite difficult to extract high-quality RNA from bulbs

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  • Method for detecting lily seed virus
  • Method for detecting lily seed virus
  • Method for detecting lily seed virus

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Experimental program
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Embodiment 1 3

[0030] Embodiment 1 Triple compound RT-MPCR technology detects lily bulb virus CMV, LSV and LMoV

[0031] 1) Use Premier Primer 5 software to design three pairs of specific viral primers with good compatibility, and use Oligo 6 software to evaluate and analyze the primers:

[0032]

[0033] 2)请参阅 figure 1 所示,为本发明采用改进的Invitrogen公司Trizol试剂法提取百合种球内层鳞片总RNA的凝胶电泳图,操作步骤如下:

[0034] (1)取-70℃保存的鳞茎内侧鳞片0.1~0.5g,在经0.1%DEPC的无菌水处理、干热灭菌后的研钵中加液氮充分研磨;

[0035] (2)加入1mLTrizol提取液,室温放置5min,使其充分裂解,磨至匀浆,12,000rpm离心15min,弃沉淀,收集提取液于离心管;

[0036] (3)加入0.2mL三氯甲烷并上下倒置混匀,12,000rpm,4℃离心20min;

[0037] (4)小心收集上清,转移至新离心管中,加入与上清等体积的异丙醇,充分混匀,室温静置10min;

[0038] (5)4℃,12,000rpm,离心10min,弃上清,RNA沉于管底;

[0039] (6)重复步骤(3)-步骤(5)3次;

[0040] (7)加入1mL75%乙醇(DEPC水配制)洗涤沉淀,4℃,7500rpm,离心10min,弃上清;

[0041] (8)沉淀干燥后,溶于20mlLRNA溶解液或DEPC水中,置于-70℃冰箱备用;或

[0042] see figure 2 所示,为本发明采用改进的CTAB法提取百合种球中层、外层鳞片RNA的凝胶电泳图,操作步骤如下:

[0043] (1)将-70℃保存的鳞片样品0.1g,在经0.1%DEPC的无菌水处理、干热灭菌后的研钵中加液氮充分研磨至粉末;

[0044] (2)迅速转入1.5mL离心管中,...

Embodiment 2

[0060] 实施例2 三重复合RT-PCR有效体系的优化

[0061] 为了优化本发明的RT-MPCR体系,以上述建立的RT-MPCR体系为基础,对其RT体系中的成份及浓度、PCR体系中的成份及浓度进行了研究。优化PCR体系的基本原则是:获得清晰、稳定的目标特异产物;成本较低;反应时间较短等。

[0062] 对RT主要成分dNTPs、三对引物,RNasin,M-MLV,以及病毒RNA浓度各设6组梯度处理。对PCR主要反应成分dNTPs,Mg 2+ ,三对引物,TaqDNA聚合酶,模板cDNA浓度以及扩增时间各设6组梯度处理。阴性对照采用建立起来的有效体系中的相应浓度。

[0063] (1)M-MLV反转录酶浓度对RT-PCR的影响

[0064] M-MLV反转录酶是RT反应体系中的关键因子之一,本实施例在RT有效体系的基础上设定了10U / μL,15U / μL,20U / μL,25U / μL,30U / μL,35U / μL等6个梯度分别进行RT反应。

[0065] (2)TagDNA聚合酶浓度对PCR扩增的影响

[0066] 酶量与PCR产物量密切相关,而且价格昂贵。本实施例在有效PCR反应体系的基础上对TagDNA聚合酶浓度设了如下处理:0.1U / μL,0.2U / μL,0.3U / μL,0.4U / μL,0.5U / μL,0.6U / μL,以期筛选其最佳低端用量。

[0067] (3)引物浓度对RT的影响

[0068] PCR体系中引物浓度通常为0.1μmol / L~1.0μmol / L,更高浓度可能导致异位引导,出现非靶序列扩增。本实施例对引物浓度设了如下处理:0.2pmol / uL,0.3pmol / uL,0.4pmol / uL,0.5pmol / uL,0.6pmol / uL,0.7pmol / uL等6个梯度。

[0069] (4)d NTPs浓度对RT-PCR的影响

[0070] 对RT中d NTPs的最佳浓度进行了研究,设定了0.5mmol / L,0.6mmol / L,0.7mmol / L,0.8mmol / L,0.9mmol / L,1.0mmol / L等6个梯度,对PCR体系设定d NTPs为0.1mmol / L,0.2mmol / L,0.3mmol / L,0.4mmol / L,0.5mmol / L,0.6mmol / L等6个梯度。

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Abstract

The invention provides a method for detecting lily seed virus including the detection of three virus of CMV, LSV and LMoV. The method comprises the following steps: (1) designing three pairs of virus primers with good compatibility; (2) adopting improved Trizol and CTAB method to extract seedball squama RNA; and (3) using RT-MPCR technique to detect virus. The method for detecting lily seed virusis stable and fast, has good repetitiveness and low cost, and can be applied to detect the CMV, LSV and LMoV virus of the lily seedball.

Description

technical field [0001] The invention relates to a detection method for lily bulb virus, in particular to a detection method for lily bulb virus CMV, LSV and LMoV. Background technique [0002] Application of Multiplex RT-PCR Technology (RT-MPCR) in Lily: Wang Jihua et al. ) detected LSV and LMoV viruses using double PCR technology, Li Haoyan et al. , 32(6), 2006, 42~45) used multiplex RT-PCR to detect LXV, LSV and LMoV, but neither of them detected CMV, and CMV virus is one of the three major viruses infecting lily, which is seriously harmful , the incidence is widespread and needs to be detected. [0003] Xu Rongxue (Xu Rongxue. Molecular detection and detoxification technology research on main viruses of lily. Nanjing Forestry University, 2007) detected the viruses in the leaves and flowers of cultivated varieties and wild species. The leaves, flowers and scales were extracted with a kit. The extraction quality of total RNA from bulbs is not high, and no RNA extraction ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12N15/10C12R1/94
Inventor 吕英民郑丽娜程堂仁张强英张启翔
Owner BEIJING FORESTRY UNIVERSITY
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