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

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 de...

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Published inJournal of agricultural and food chemistry Vol. 65; no. 38; pp. 8302 - 8310
Main Authors Qian, Wenjuan, Meng, Youqing, Lu, Ying, Wu, Cui, Wang, Rui, Wang, Liu, Qian, Cheng, Ye, Zunzhong, Wu, Jian, Ying, Yibin
Format Journal Article
LanguageEnglish
Published United States American Chemical Society 27.09.2017
Subjects
Citrus - chemistry
Citrus - microbiology
Luminescent Measurements - methods
Nucleic Acid Amplification Techniques - methods
Organic Chemicals - chemistry
Plant Diseases - microbiology
Plant Leaves - chemistry
Plant Leaves - microbiology
Rhizobiaceae - chemistry
Rhizobiaceae - genetics
Rhizobiaceae - isolation & purification
crude DNA extraction
huanglongbing
Candidatus Liberibacter asiaticus
visual detection
field detection
isothermal amplification
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Abstract 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.
AbstractList 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.
Author Wang, Liu
Lu, Ying
Wu, Jian
Wu, Cui
Qian, Wenjuan
Meng, Youqing
Qian, Cheng
Ying, Yibin
Ye, Zunzhong
Wang, Rui
AuthorAffiliation College of Biosystems Engineering and Food Science
Zhejiang A&F University
Key Laboratory of On Site Processing Equipment for Agricultural Products
Zhejiang Plant Protection and Quarantine Bureau
Ministry of Agriculture
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Keywords crude DNA extraction
huanglongbing
Candidatus Liberibacter asiaticus
visual detection
field detection
isothermal amplification
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Snippet Huanglongbing is a devastating citrus disease, and ‘Candidatus Liberibacter asiaticus’ (Las) is the most prevalent huanglongbing-associated bacterium. Its...
Huanglongbing is a devastating citrus disease, and 'Candidatus Liberibacter asiaticus' (Las) is the most prevalent huanglongbing-associated bacterium. Its...
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SubjectTerms Citrus - chemistry
Citrus - microbiology
Luminescent Measurements - methods
Nucleic Acid Amplification Techniques - methods
Organic Chemicals - chemistry
Plant Diseases - microbiology
Plant Leaves - chemistry
Plant Leaves - microbiology
Rhizobiaceae - chemistry
Rhizobiaceae - genetics
Rhizobiaceae - isolation & purification
Title Rapid, Sensitive, and Carryover Contamination-Free Loop-Mediated Isothermal Amplification-Coupled Visual Detection Method for ‘Candidatus Liberibacter asiaticus’
URI http://dx.doi.org/10.1021/acs.jafc.7b03490
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