Qian Wenjuan, Meng Youqing, Lu Ying, Wu Cui, Wang Rui, Wang Liu, Qian Cheng, Ye Zunzhong, Wu Jian, Ying Yibin
College of Biosystems Engineering and Food Science, Zhejiang University , Hangzhou, Zhejiang 310058, People's Republic of China.
Key Laboratory of On Site Processing Equipment for Agricultural Products, Ministry of Agriculture , Hangzhou, Zhejiang 310058, People's Republic of China.
J Agric Food Chem. 2017 Sep 27;65(38):8302-8310. doi: 10.1021/acs.jafc.7b03490. Epub 2017 Sep 13.
Huanglongbing is a devastating citrus disease, and 'Candidatus Liberibacter asiaticus' (Las) is the most prevalent huanglongbing-associated bacterium. Its field detection remains challenging. In this work, a visual, rapid, sensitive, and carryover contamination-free method was developed for field detection of Las. Leaf samples were treated with 500 μL of 0.5 M sodium hydroxide solution for 3 min, and 50-fold dilutions were directly amplified by loop-mediated isothermal amplification. Then, a novel SYTO-9-based visual detection method was used to evaluate amplification results without uncapping operation. Negative samples remained colorless, while positive samples generated obvious green fluorescence, which could be easily distinguished by the naked eye with a mini-fluorescent-emission cartridge developed originally. The proposed detection method could be accomplished within 40 min and is about 100 times more sensitive than conventional TaqMan polymerase chain reaction. The reliability of this method was also verified by analyzing practical samples.
黄龙病是一种极具毁灭性的柑橘病害，“亚洲韧皮杆菌”（Las）是与黄龙病关联最为普遍的细菌。其田间检测仍然具有挑战性。在这项工作中，开发了一种用于田间检测Las的可视化、快速、灵敏且无残留污染的方法。叶片样本用500 μL 0.5 M氢氧化钠溶液处理3分钟，50倍稀释液通过环介导等温扩增直接进行扩增。然后，使用一种基于新型SYTO-9的可视化检测方法来评估扩增结果，无需开盖操作。阴性样本保持无色，而阳性样本产生明显的绿色荧光，使用最初开发的微型荧光发射盒，肉眼即可轻松区分。所提出的检测方法可在40分钟内完成，灵敏度比传统TaqMan聚合酶链反应高约100倍。通过分析实际样本也验证了该方法的可靠性。
