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Rapid, Sensitive, and Carryover Contamination-Free Loop-Mediated Isothermal Amplification-Coupled Visual Detection Method for ‘Candidatus Liberibacter asiaticus’

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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
§ Zhejiang Plant Protection and Quarantine Bureau, Hangzhou, Zhejiang 310020, People’s Republic of China
Zhejiang A&F University, Hangzhou, Zhejiang 311300, People’s Republic of China
*Telephone/Fax: 0086-571-88982282. E-mail: [email protected]
Cite this: J. Agric. Food Chem. 2017, 65, 38, 8302–8310
Publication Date (Web):August 31, 2017
https://doi.org/10.1021/acs.jafc.7b03490
Copyright © 2017 American Chemical Society

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    Abstract

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    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.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b03490.

    • Regents, standard DNA extraction, specificity of real-time LAMP, LAMP-coupled visual detection method, and real-time TaqMan PCR (Table S1), detection results of 30 asymptomatic and 30 symptomatic leaf samples by real-time LAMP and real-time TaqMan PCR (Table S2), detection result of 352 field samples in Zhejiang province (Table S3), comparisons between Tt values for LAMP and Ct values for TaqMan PCR of the positive results in 352 field samples (Table S4), LAMP amplification components for visual detection (Table S5), and detection result of 30 asymptomatic leaf samples by the LAMP-coupled visual detection method (Figure S1) (PDF)

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