Dai Tingting, Hu Tao, Yang Xiao, Shen Danyu, Jiao Binbin, Tian Wen, Xu Yue
Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, Jiangsu, China.
United States Department of Agriculture, Agricultural Research Service, Foreign Disease-Weed Science Research Unit, Fort Detrick, MD, USA.
PeerJ. 2019 Nov 18;7:e8083. doi: 10.7717/peerj.8083. eCollection 2019.
, the causal agent of brown rot of citrus fruit, is an important worldwide pathogen and a quarantine pest in China. Current diagnosis of the disease relies on disease symptoms, pathogen isolation and identification by DNA sequencing. However, symptoms caused by can be confused with those by other and fungal species. Moreover, pathogen isolation, PCR amplification and sequencing are time-consuming. In this study, a rapid assay including 20-min recombinase polymerase amplification targeting the gene and 5-min visualization using lateral flow dipsticks was developed for detecting . This assay was able to detect 0.2 ng of genomic DNA in a 50-µL reaction system. It was specific to without detection of other tested species including , , and , four other important citrus pathogens. Using this assay, was also detected from artificially inoculated orange fruits. Results in this study indicated that this assay has the potential application to detect at diagnostic laboratories and plant quarantine departments of customs, especially under time- and resource-limited conditions.
柑橘褐腐病菌是柑橘类水果褐腐病的病原体，是一种在全球范围内重要的病原菌，在中国属于检疫性有害生物。目前该病的诊断依赖于病害症状、病原菌分离以及通过DNA测序进行鉴定。然而，柑橘褐腐病菌引起的症状可能与其他细菌和真菌物种引起的症状相混淆。此外，病原菌分离、PCR扩增和测序都很耗时。在本研究中，开发了一种快速检测方法，包括针对柑橘褐腐病菌基因进行20分钟的重组酶聚合酶扩增以及使用侧向流动试纸条进行5分钟的可视化检测，用于检测柑橘褐腐病菌。该检测方法能够在50微升反应体系中检测到0.2纳克的柑橘褐腐病菌基因组DNA。它对柑橘褐腐病菌具有特异性，未检测到其他受试物种，包括另外四种重要的柑橘病原菌——柑橘溃疡病菌、柑橘黄龙病菌、柑橘炭疽病菌和柑橘链格孢菌。使用该检测方法，还从人工接种的橙子果实中检测到了柑橘褐腐病菌。本研究结果表明，该检测方法在诊断实验室以及海关植物检疫部门检测柑橘褐腐病菌方面具有潜在应用价值，尤其是在时间和资源有限的条件下。
