甘薯羽状斑驳病毒RT-LAMP快速检测方法的建立

doi:10.3864/j.issn.0578-1752.2018.07.007

摘要/Abstract

摘要： 【目的】甘薯羽状斑驳病毒（Sweet potato feathery mottle virus，SPFMV）侵染甘薯造成重要危害，本研究旨在利用反转录环介导等温扩增技术（reverse transcription loop-mediated isothermal amplification，RT-LAMP）建立一种快速、高效检测SPFMV的方法。【方法】从GenBank上获得SPFMV外壳蛋白（coat protein，CP）基因的核苷酸序列，利用Primer Explorer V4设计4条RT-LAMP特异性引物SPFMV-FIP（5′-TAAGCGCGGCTGCC TTCATC-CATTCAACCACCCCTGCA-3′）、SPFMV-BIP（5′-TCGGTTGTTTGGTTTGGACGGA-ATCAGTTGTCGTGTGCCTC-3′）、SPFMV-F3（5′-GAGTCTTGCGCGATATGCA-3′）和SPFMV-B3（5′-ACCCCTCATTCCTAAGAGGT-3′），同时设计2条反转录聚合酶链反应（reverse transcription polymerase chain reaction，RT-PCR）的特异性引物SPFMV-F（5′-TCTAATGAGAACACTGAA TT-3′）和SPFMV-R（5′-TTGCACACCCCTCATTCCTAAG-3′）。分别设置F3/B3﹕FIP/BIP引物浓度比（1﹕1、1﹕2、1﹕4、1﹕6、1﹕8和1﹕10），dNTPs浓度梯度（0.025、0.125、0.225、0.325、0.425、0.525、0.625、0.725和0.825 mmol·L^-1），Betaine浓度梯度（0.4、0.7、1.0、1.3和1.6 mol·L^-1），反应温度（59、61、63、65、67和69℃）和反应时间（20、30、40、50、60、70、80和90 min），对RT-LAMP反应体系各条件进行优化，确定最佳反应体系。通过测序及酶切对RT-LAMP产物进行鉴定。以携带SPFMV、甘薯C病毒（Sweet potato virus C，SPVC）、甘薯褪绿矮化病毒（Sweet potato chlorotic stunt virus，SPCSV）、甘薯病毒2（Sweet potato virus 2，SPV2）、甘薯潜隐病毒（Sweet potato latent virus，SPLV）、甘薯G病毒（Sweet potato virus G，SPVG）、甘薯褪绿斑病毒（Sweet potato chlorotic fleck virus，SPCFV）和健康甘薯叶片的总RNA为模板，分别进行RT-LAMP和RT-PCR特异性检测；带有SPFMV的甘薯总RNA进行10倍梯度稀释，以RNA原液、10^-1、10^-2、10^-3、10^-4、10^-5、10^-6和10^-7稀释液为模板进行RT-LAMP和RT-PCR灵敏度测定。最后利用优化的RT-LAMP体系对山东省多地采集的SPFMV疑似病样进行检测，加入SYBR green I进行可视化检测。【结果】建立了SPFMV的RT-LAMP快速特异性检测方法，优化的反应体系：引物SPFMV-FIP/SPFMV-BIP为0.8 µmol·L^-1，SPFMV-F3/SPFMV-B3为0.2 µmol·L^-1，dNTPs为0.325 mmol·L^-1，Betaine为1 mol·L^-1；65℃反应70 min。特异性试验显示本研究建立的RT-LAMP只对携带SPFMV的RNA能够扩增出典型的梯状条带。RT-LAMP最低可检测的RNA浓度为121.6×10^-4 ng·μL^-1，而RT-PCR最低能检测的RNA浓度为121.6×10^-3 ng·μL^-1，表明RT-LAMP的灵敏度比RT-PCR高10倍。田间样品检测，RT-LAMP扩增结果与可视化检测结果一致，表明本研究建立的RT-LAMP快速检测方法可有效应用到SPFMV的田间检测。【结论】建立的RT-LAMP快速检测方法灵敏度高、特异性好，适用于SPFMV的田间快速检测。

关键词: 甘薯, 甘薯羽状斑驳病毒, 反转录环介导等温扩增技术, 检测

Abstract: 【Objective】Sweet potato feathery mottle virus (SPFMV) is an important virus infecting sweet potato plants. The objective of this study is to establish a rapid and efficient method for the detection of SPFMV by using reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. 【Method】Four specific RT-LAMP primers for SPFMV detection including SPFMV-FIP (5′-TAAGCGCGGCTGCCTTCATC-CATTCAACCACCCCTGCA-3′), SPFMV-BIP (5′-TCGGTTGTTTGGT TTGGACGGA-ATCAGTTGTCGTGTGCCTC-3′), SPFMV-F3 (5′-GAGTCTTGCGCGATATGCA-3′) and SPFMV-B3 (5′-ACCCC TCATTCCTAAGAGGT-3′) were designed by Primer Explorer V4 according to the nucleotide sequence of SPFMV coat protein (CP) gene in GenBank as well as two specific reverse transcription polymerase chain reaction (RT-PCR) primers for SPFMV detection including SPFMV-F (5′-TCTAATGAGAACACTGAATT-3′) and SPFMV-R (5′-TTGCACACCCCTCATTCCTAAG-3′). Different reaction conditions were optimized in the RT-LAMP in order to improve specificity and sensitivity of the detection, including the primers concentration ratios of F3/B3 to FIP/BIP (1﹕1, 1﹕2, 1﹕4, 1﹕6, 1﹕8 and 1﹕10), dNTPs concentrations (0.025, 0.125, 0.225, 0.325, 0.425, 0.525, 0.625, 0.725 and 0.825 mmol·L^-1), Betaine concentrations (0.4, 0.7, 1.0, 1.3 and 1.6 mol·L^-1), reaction temperatures (59, 61, 63, 65, 67 and 69℃) and reaction times (20, 30, 40, 50, 60, 70, 80 and 90 min). The best reaction conditions were confirmed according to the test results of agarose gel electrophoresis. The RT-LAMP products were identified by sequencing and enzyme analysis. The detection specificity of RT-LAMP was tested by using different RNA templates from SPFMV, Sweet potato virus C (SPVC), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus 2 (SPV2), Sweet potato latent virus (SPLV), Sweet potato virus G (SPVG), Sweet potato chlorotic fleck virus (SPCFV) and leaf sample of healthy sweet potato plant. The sensitivities of RT-LAMP and RT-PCR for detecting SPFMV were compared by using ten-fold serially diluted RNA templates of SPFMV (including original RNA, 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6 and 10^-7 dilutions). Finally, the optimized RT-LAMP and RT-PCR were used to detect the samples of SPFMV collected from Shandong Province. 【Result】The RT-LAMP rapid detection method of SPFMV was established and the optimal amplification was achieved by incubation of 0.8 µmol·L^-1 SPFMV-FIP/SPFMV-BIP, 0.2 µmol·L^-1 SPFMV-F3/SPFMV-B3, 0.325 mmol·L^-1 dNTPs, 1 mol·L^-1 Betaine with template RNA at 65℃ for 70 min. The specificity test showed that the RT-LAMP method established in this study could amplify the typical ladder-like bands only to the RNA carrying SPFMV. The lowest detectable RNA concentration of RT-LAMP was 121.6×10^-4 ng·μL^-1, while the lowest detectable RNA concentration of RT-PCR was 121.6×10^-3 ng·μL^-1, indicating that the sensitivity of RT-LAMP was 10 times higher than RT-PCR for detecting SPFMV. The optimized RT-LAMP method was applied to the detection of SPFMV in field samples and the results were consistent with the visual inspection of RT-LAMV products. It suggested that the RT-LAMP detection method could be applied to detect the SPFMV in the field.【Conclusion】The RT-LAMP established in this study has high sensitivity and speci?city, and is suitable for rapid detection of SPFMV in the field.

Key words: sweet potato; Sweet potato feathery mottle virus (SPFMV), RT-LAMP, detection

姜珊珊，冯佳，张眉，王升吉，辛志梅，吴斌，辛相启. 甘薯羽状斑驳病毒RT-LAMP快速检测方法的建立[J]. 中国农业科学, 2018, 51(7): 1294-1302.

JIANG ShanShan, FENG Jia, ZHANG Mei, WANG ShengJi, XIN ZhiMei, WU Bin, XIN XiangQi. Development of RT-LAMP Assay for Rapid Detection of Sweet potato feathery mottle virus (SPFMV)[J]. Scientia Agricultura Sinica, 2018, 51(7): 1294-1302.

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