草莓轻型黄边病毒RT-LAMP检测方法的建立

doi:10.3864/j.issn.0578-1752.2015.03.20

摘要/Abstract

摘要： 【目的】草莓轻型黄边病毒（Strawberry mild yellow edge virus，SMYEV）是侵染草莓的重要病毒，严重影响草莓果实的产量和品质。研究旨在建立利用反转录等温扩增技术（reverse transcription loop-mediated isothermal amplification,RT-LAMP）检测SMYEV的方法。【方法】根据SMYEV外壳蛋白基因3′端保守序列设计4个特异性引物SMYEV-FIP（5′-CAGATCAGCGACAATTTGGACTCCTGAGGAACTTGCTGCT-3′）、SMYEV-BIP（5′-GCTTTGTC GGGGATCCTGGGAAGGCTAAGTCGAAGAGACC-3′）、SMYEV-F₃（5′-TCAAGTTGGTGACCCTTTCC-3′）和SMYEV-B₃（5′-CGAGGAAC CAATGTCGTAGC-3′）。对RT-LAMP 检测体系中的条件进行优化，设置反应温度为60、61、62、63、64、65℃，反应时间为 30、45、60、75 min，分别设置引物SMYEV-FIP/BIP浓度为1.0、1.2、1.4、1.6、1.8 µmol?L^-1，引物SMYEV-F3/B3浓度0.1、0.15、0.2、0.25、0.3 µmol?L^-1，设置Mg²⁺浓度为2、4、6、8、10 mmol?L^-1，dNTPs浓度为0、0.4、0.8、1.2、1.6、2.0 mmol?L^-1，betaine浓度为0、0.4、0.8、1.0、1.2、1.4 mol?L^-1，DTT浓度为2.0、2.4、2.8、3.2、3.6、4.0 µmol?L^-1等，并对以上不同反应条件进行检测，确定优化后的RT-LAMP检测体系。选用其他常见的草莓病毒以及健康草莓植株叶片中的RNA作为模板，对RT-LAMP体系的特异性进行测定；将SMYEV的RNA进行10倍梯度逐级稀释，分别获得RNA原液、10^-1、10^-2、10^-3、10^-4、10^-5、10^-6和10^-7不同稀释液作为模板，比较RT-LAMP 和RT-PCR的检测灵敏度。RT-LAMP反应产物可以进行电泳和紫外成像检测，阳性样品出现瀑布状条带，阴性样品无条带，或者在反应产物中加入SYBR green I核酸染料，直接观察检测，阳性样品为绿色，阴性样品为橙色。【结果】建立了一种特异性检测SMYEV的RT-LAMP方法，经过各反应条件优化后的检测体系为1.0 µmol?L^-1 SMYEV-FIP/BIP、0.1 µmol?L^-1SMYEV-F₃/B₃、4 mmol?L^-1 Mg²⁺、1.6 mmol?L^-1 dNTPs、0.4 mol?L^-1 betaine、2.0 µmol?L^-1 DTT，60℃反应45 min。选用SMYEV、草莓镶脉病毒（Strawberry vein banding virus，SVBV）、草莓斑驳病毒（Strawberry mottle virus，SMoV）和草莓皱缩病毒（Strawberry crinkle virus，SCV）的RNA以及健康草莓植株叶片中的RNA作为模板进行RT-LAMP的特异性检测，结果显示仅有SMYEV的RNA能够扩增出瀑布型条带，建立的RT-LAMP检测体系有很好的特异性。将SMYEV的RNA原液、10^-1、10^-2、10^-3稀释液作为模板进行RT-PCR反应后，检测结果为阳性，随着模板稀释倍数的增加，电泳检测不能观察到RT-PCR的扩增产物。而进行SMYEV的RT-LAMP检测时，RNA原液、10^-1、10^-2、10^-3、10^-4和10^-5稀释液作为模板时，均可以观察到阳性结果。RT-LAMP检测SMYEV比RT-PCR方法检测的灵敏度高100倍，并且比RT-PCR节省时间，检测结果容易判定。【结论】RT-LAMP方法能快速、简便、特异的检测SMYEV，供科研部门和基层生产单位使用，在种苗繁育、田间调查和海关检疫的过程中进行快速检测。

关键词: 草莓轻型黄边病毒, 反转录等温扩增技术, 检测

Abstract: 【Objective】Strawberry mild yellow edge virus (SMYEV) is an important virus infecting strawberry plants, reducing fruit yield and quality. The objective of this study is to establish an effective method to detect SMYEV using the reverse transcription loop-mediated isothermal amplification (RT-LAMP). 【Method】 Four specific RT-LAMP primers for SMYEV detection including SMYEV-FIP (5′-CAGATCAGCGACAATTTGGACTCCTGAGGAACTTGCTGCT-3′), SMYEV-BIP (5′-GCTTTGTCGGGGATC CTGGGAAGGCTAAGTCGAAGAGACC-3′), SMYEV-F3 (5′-TCAAGTTGGTGACCCTTTCC-3′) and SMYEV-B3 (5′-CGAGG AACCAATGTCGTAGC-3′) were designed according to the published 3′ end conservative sequences of SMYEV CP gene. Different reaction temperatures (60, 61, 62, 63, 64, 65℃), reaction times (30, 45, 60, 75 min), concentrations of primers SMYEV-FIP/BIP (1.0, 1.2, 1.4, 1.6, 1.8 µmol?L^-1) and SMYEV-F3/B3 (0.1, 0.15, 0.2, 0.25, 0.3 µmol?L^-1), Mg²⁺(2, 4, 6, 8, 10 mmol?L^-1), dNTPs (0, 0.4, 0.8, 1.2, 1.6, 2.0 mmol?L^-1), betaine (0, 0.4, 0.8, 1.0, 1.2, 1.4 mol?L^-1) and DTT (2.0, 2.4, 2.8, 3.2, 3.6, 4.0 µmol?L^-1) were used and optimized in the RT-LAMP in order to improve specificity and sensitivity of the detection. The specificity of RT-LAMP was tested by using different RNA templates from other important strawberry viruses and healthy leaves of strawberry plants. The sensitivities of RT-LAMP and RT-PCR for detecting SMYEV were compared by using ten-fold serially diluted RNA templates of SMYEV (including original RNA, 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6and 10^-7 dilution). The RT-LAMP products could be detected by electrophoresis and ultraviolet image technology, the typical ladder-like pattern was observed in the positive samples while no amplification of DNA was visible in the negative samples. The RT-LAMP products could be also evaluated by adding SYBR green I, the color of the product for the positive reaction changed from orange to green while the product for a negative reaction remained orange.【Result】The specific RT-LAMP method to detect SMYEV was established and the optimal amplification was achieved by incubation of 1.0 µmol?L^-1SMYEV-FIP/BIP, 0.1 µmol?L^-1 SMYEV-F₃/B₃, 4 mmol?L^-1 Mg²⁺, 1.6 mmol?L^-1 dNTPs, 0.4 mol?L^-1 betaine, 2.0 µmol?L^-1 DTT with template RNA at 60℃ for 45 min. The detection specificity of RT-LAMP was tested by using different RNA templates from SMYEV, Strawberry vein banding virus, Strawberry mottle virus, Strawberry crinkle virus and leaf sample of healthy strawberry plant. The optimized RT-LAMP method had high sensitivity that only reaction within SMYEV RNA template could produce typical ladder-like bands tested by electrophoresis and UV. Test results of RT-PCR detection were positive only with original and 10^-1, 10^-2, 10^-3 diluent RNA templates and no amplified band could be detected when using diluent RNA template with the increase of dilution ratio. While in RT-LAMP detection positive results could be observed with original, 10^-1, 10^-2, 10^-3, 10^-4, and 10^-5 diluent RNA templates. Sensitivity of the RT-LAMP was 100 times higher than the RT-PCR method for detecting SMYEV. It was time-saving and the results could be directly observed in RT-LAMP which are suitable for SMYEV detection.【Conclusion】 The optimized RT-LAMP proved to be a faster, simpler and specific method for SMYEV detection and could be applied in the process of seedling breeding, field investigation and customs quarantine control in both research institutions and rural areas.

Key words: Strawberry mild yellow edge virus; RT-LAMP, detection

陈柳，尚巧霞，陈笑瑜，邢冬梅，冉策，魏艳敏，赵晓燕，刘正坪. 草莓轻型黄边病毒RT-LAMP检测方法的建立[J]. 中国农业科学, 2015, 48(3): 613-520.

CHEN Liu, SHANG Qiao-xia, CHEN Xiao-yu, XING Dong-mei, RAN Ce, WEI Yan-min, ZHAO Xiao-yan, LIU Zheng-ping. Detection of Strawberry mild yellow edge virus by RT-LAMP[J]. Scientia Agricultura Sinica, 2015, 48(3): 613-520.

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