Abstract: 【Objective】The objective of this study is to establish a method which uses reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology, and to detect Apple chlorotic leaf spot virus (ACLSV) simply and quickly. 【Method】Three sets of specific primers were designed based on conserved region of ACLSV genomes. Each set of primers includes a pair of outer primer (F3/B3) and a pair of inner primer (FIP/BIP). One feasible set of primers was selected for the RT-LAMP reaction. RT-LAMP reaction system was optimized, that is, the concentration of Mg²⁺ (0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0 mmol·L^-1), dNTPs (0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 mmol·L^-1), Betaine (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mol·L^-1), FIP/BIP (0.8, 1.2, 1.6, 2.0, 2.4 µmol·L^-1) and F3/ B3 (0, 0.1, 0.2, 0.3, 0.4 µmol·L^-1), respectively. The RT-LAMP reaction condition was optimized, using optimized reaction system, reaction temperature was set to 65, 63, 61, 59, 57℃ and reaction time was set for 90 min. In the process of primers screening and optimizing the reaction system, fluorescent pigment was added to the reaction system and real-time PCR instrument was used. The whole process was detected in real-time by fluorescence signal accumulation. Finally, the amplification curve was used to analyze the reaction result. the specificity detection of RT-LAMP was tested by using different RNA templates of infected leaves from ACLSV, Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV) and Apple mosaic virus (ApMV). To assess the detection sensitivity, 10⁰, 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6 diluents of original RNA were used as templates of RT-LAMP. To evaluate the application value of this method, 23 apple leaf samples were collected randomly in the field, the optimized RT-LAMP and RT-PCR were used to detect the samples, SYBR GreenⅠwas added to visualize the detection. 【Result】 The RT-LAMP method to detect ACLSV was established. The optimized detection system was 6.0 mmol·L^-1 Mg²⁺, 1.2 mmol·L^-1 dNTPs, 0.2 mol·L^-1 Betaine, 1.6 µmol·L^-1 FIP/BIP and 0.2 µmol·L^-1 F3/B3 primer. The optimum reaction conditions were 59℃ and 60 min. In the specificity test, only the ACLSV test result was positive, the controls were all negative. In the sensitivity test, the 10^-3 RNA dilution could be detected by RT-LAMP method. It was 100 times higher sensitivity than RT-PCR method. The positive rate of RT-PCR of randomly selected 23 apple leaf samples was 52.2%, and RT-LAMP was 65.2%. Detection rate of RT-LAMP was higher than that of RT-PCR. 【Conclusion】The established ACLSV RT-LAMP detection method is simple, quick, sensitive and low cost. It can be applied in field investigation, seeding breeding and customs quarantine.

Key words: apple, Apple chlorotic leaf spot virus (ACLSV), reverse transcription loop-mediated isothermal amplification (RT-LAMP), real-time PCR, virus detection