Abstract:

【Background】 Citrus Huanglongbing (HLB) is a citrus disease caused by ‘Candidatus Liberibacter asiaticus’ (CLas). The main approaches to control HLB include plant quarantine, establishing disease-free nurseries, removing disease trees, and concentrating on large area joint control of citrus psyllids (Diaphorina citri). The first three methods all rely on accurate HLB diagnosis techniques.【Objective】 The objective of this study is to establishment of a rapid and handy field/laboratory nucleic acid detection method of CLas using loop-mediated isothermal amplification (LAMP) combined with membrane adsorption rapid DNA extraction and Gelgreen fluorescence dye visualization.【Method】 The LAMP primers were designed using the β-operon and the prophage DNA polymerase gene of CLas as templates, including outer primer F3/B3, inner primer FIP/BIP, loop primer LoopF/LoopB and stem primer StemF/StemB. The LAMP primer set was optimized by setting different dosage combinations for loop primers and stem primers to determine the appropriate primer concentration. A total of 188 field citrus leaves were detected using the optimized LAMP primer set, and the receiver operating characteristic (ROC) curves were constructed to analyze the accuracy of real-time fluorescent LAMP (qLAMP) for CLas detection. The qLAMP premixed reaction solution was dried in two steps at room temperature, storage temperature (4, 25 and 35℃) and storage time (1, 2 and 4 weeks) were also set to assess the enzyme activity stability of the dry LAMP reagent. Using dry LAMP reagent, combined with membrane adsorption rapid DNA extraction technique in this study, 71 citrus leaf samples and 35 citrus fruit samples collected in the field were detected, while the detection results of real-time fluorescent quantitative (qPCR) were used as controls to compare the coincidence rates of the two detection methods.【Result】 The addition of loop primer, stem primer or increasing their concentrations in LAMP reaction could promote the increase of reaction rate, and the addition of both loop primer and stem primer at a final concentration of 1.6 μmol·L^-1 could further improve the reaction rate. The reaction activity of LAMP premix could be maintained unchanged by two-step drying at different temperatures for 1-4 weeks, indicating that the two-step drying LAMP reagent prepared in this experiment had good detection performance and fair stability at low and room temperatures, and only at 35℃ storage would slightly increase the reaction time of LAMP reagent. Using 0.1 μm pore size nylon membrane instead of cellulose filter paper as nucleic acid adsorption material could improve the sensitivity of rapid diagnostic techniques. The overall accuracy of rapid DNA diagnosis for HLB established by combining rapid DNA extraction and visual LAMP was high, and the lowest detectable plasmid concentration was 10² copies/μL. The diagnostic results of this method were not significantly different from those of qPCR by paired Chi-square test. The visual LAMP rapid detection was less cost and time-consuming than routine detection, and visual LAMP rapid detection required no expensive instruments such as centrifuges and PCR instruments, requiring only a 65℃ thermostatic device.【Conclusion】 The rapid DNA detection method for CLas established in this study has low cost and can observe detection results in 30 min, easy to operate and high accuracy, which can replace qPCR for rapid identification of HLB in the field.

Key words: citrus Huanglongbing, ‘Candidatus Liberibacter asiaticus’ (CLas), field detection, membrane adsorption, rapid DNA extraction, visual LAMP