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