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高彦萍 1, 3, 张 武 1, 3, 王国祥 2, 席春艳 1, 吴雁斌 1, 3, 梁宏杰 1, 3, 吕和平 1, 3*.马铃薯卷叶病毒PLRV RT-LAMP检测方法优化[J].植物保护,2019,45(6):259-264.
马铃薯卷叶病毒PLRV RT-LAMP检测方法优化
Optimization of the PLRV RT-LAMP detection method
投稿时间:2018-11-23  修订日期:2019-02-17
DOI:10.16688/j.zwbh.2018484
中文关键词:  马铃薯卷叶病毒(PLRV)  反转录环介导等温扩增(RT-LAMP)  检测方法
英文关键词:Potato leafroll virus(PLRV)  RT-LAMP  reaction system
基金项目:甘肃省农业生物技术研究与应用开发项目(GNSW-2016-15); 国家重点研发计划(2017YFD0201602-4, 2018YFD020080501); 甘肃省农业科学院院列项目(2019GAAS04, 2017GAAS29, 2017GAAS90)
作者单位E-mail
高彦萍 1, 3, 张 武 1, 3, 王国祥 2, 席春艳 1, 吴雁斌 1, 3, 梁宏杰 1, 3, 吕和平 1, 3* 1. 甘肃省农业科学院马铃薯研究所, 兰州 730070
2. 甘肃省农业科学院中药材研究所, 兰州 730070
3. 甘肃省马铃薯脱毒种薯种苗病毒检测及安全评价工程技术研究中心, 兰州 730070 
lheping@qq.com 
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中文摘要:
      马铃薯卷叶病毒Potato leafroll virus (PLRV)是目前严重影响马铃薯产量与品质的主要病毒之一, 给马铃薯产业造成巨大损失。本研究采用环介导等温核酸扩增(loop-mediated isothermal amplification, LAMP)技术建立PLRV的RT-LAMP检测方法。采取单因素变化试验, 对RT-LAMP反应体系中多个因素包括引物组合、温度条件及Mg 2+、betaine、Bst 3.0 DNA聚合酶、dNTPs、UNG、SYBR Green Ⅰ和引物组合的浓度进行一系列试验和优化。采用RT-PCR检测方法进行平行比对试验, 对优化后的RT-LAMP反应体系进行了验证。结果表明, 最佳引物组合为P3, 最适反应温度62℃, 25 μL反应体系中, Mg 2+、betaine、Bst 3.0 DNA聚合酶和UNG的最佳终浓度分别为4 mmol/L、0 mmol/L、0.64 U/μL和0.08 U/μL, dNTPs的最佳用量为1 μL(dATP、dGTP、dCTP各0.4 mmol/L, dUTP 1.2 mmol/L), SYBR Green Ⅰ(20×)的最佳用量1 μL, primer mix的最佳用量2.5 μL(PLRV-FIP/BIP、PLRV-F3/B3和PLRV-LF/LB的浓度分别为0.8、0.2 μmol/L和0.6 μmol/L), RNA模板1 μL(2 ng/μL), 加DEPC-H2O至25 μL, 反应时间50 min。优化后的RT-LAMP检测结果与RT-PCR一致, 且可视化判读结果。因此, 建立的PLRV RT-LAMP检测方法为进一步开发RT-LAMP检测试剂盒及其实际应用奠定了基础。
英文摘要:
      Potato leafroll virus (PLRV) is currently one of the main threats for the yield and quality of potatoes, having caused tremendous damages to the potato industry. In this study, a PLRV reverse transcription-loop mediated isothermal amplification (RT-LAMP) method was established based on the loop-mediated isothermal amplification (LAMP). Single-factor experiments were conducted to test and optimize the RT-LAMP reaction system, including the primers, temperature, Mg 2+, betaine, Bst 3.0 DNA polymerase, dNTPs, UNG, SYBR Green Ⅰ and primer mix concentrations. The optimized RT-LAMP reaction system was then verified through parallel-controlled test using the reverse transcription-polymerase chain reaction (RT-PCR) method. The results showed that, in the optimized reaction conditions, the primer pair was P3 and the reaction temperature was 62℃; in the 25 μL reaction system, the concentrations of Mg 2+, betaine Bst 3.0 DNA polymerase, and UNG were 4 mmol/L, 0 mmol/L, 0.64 U/μL, and 0.08 U/μL, respectively; the dosage for dNTPs was 1 μL (0.4 mmol/L for dATP, dGTP, and dCTP, respectively, and 1.2 mmol/L for dUTP); the dosage for SYBR Green Ⅰ(20×) was 1 μL; the dosage for the primer mix was 2.5 μL (the corresponding concentrations for PLRV-FIP/BIP, PLRV-F3/B3, and PLRV-LF/LB was 0.8 μmol/L, 0.2 μmol/L, and 0.6 μmol/L, respectively); for the 1 μL RNA template (2 ng/μL), additional DEPC-H2O was added to get a final 25 μL volume, and the reaction time was 50 min. The optimized RT-LAMP provided the same detection result as RT-PCR and the interpretation could be visualized. These results confirmed that our PLRV RT-LAMP reaction system provides a basis for further developing RT-LAMP detection kits and for their practical application.
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