中国农业科学Issue(3)：614-621,8.DOI:10.3864/j.issn.0578-1752.2015.03.20
草莓轻型黄边病毒RT-LAMP检测方法的建立
Detection ofStrawberry mild yellow edge virus by RT-LAMP
摘要
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.关键词
草莓轻型黄边病毒/反转录等温扩增技术/检测Key words
Strawberry mild yellow edge virus/RT-LAMP/detection引用本文复制引用
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,(3):614-621,8.基金项目
北京市教育委员会科技计划面上项目（KM201210020003）、青年拔尖人才培育计划 （）
