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

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.

Uracil-Mediated New Photospacer-Adjacent Motif of Cas12a To Realize Visualized DNA Detection at the Single-Copy Level Free from Contamination

“…Candidatus Liberibacter asiaticus (Las) is described as being associated with citrus HLB. , 60 diseased leaves infected with Las from 60 different citrus trees were employed as our samples. All of these samples have been tested with standard PCR assay described in our previous work. , A brief workflow is described in Figure B. The DNA extracted from a half-inch by a half-inch leaf mid-rib region was used as the template both for the experimental group with ULC assay and control group with normal LAMP.…”

“…All of these samples have been tested with standard PCR assay described in our previous work. 31,32 A brief workflow is described in Figure 4B. The DNA extracted from a half-inch by a half-inch leaf mid-rib region was used as the template both for the experimental group with ULC assay and control group with normal LAMP.…”

LAMP Diagnostics at the Point-of-Care: Emerging Trends and Perspectives for the Developer Community

“…• Loop-mediated isothermal amplification (LAMP) is an emerging nucleic acid amplification technique with potential applications at the point of care • Sample type, sample preparation, and detection modalities are important considerations during LAMP assay design • One of the greatest opportunities as well as challenges for LAMP is the possibility of performing direct amplification from crude samples without extraction • Reverse transcription (RT)-LAMP is a promising alternative to polymerase chain reaction (PCR) for rapid SARS-CoV-2 testing, including methods leveraging extraction-free amplification • An improved understanding of LAMP primer design and falsepositives along with more rigorous assay characterization is necessary to improve translation of LAMP to the point of care can aerosolize during analysis and interfere with subsequent LAMP assays. This can be prevented by separating preparation from analysis spaces or implementing closed-tube detection mechanisms [27][28][29]. Mitigation of false-positive amplification, either by iterative primer design or careful tuning of reaction conditions, is an important consideration when using LAMP for clinical diagnostics.…”

Dual-color blending based visual LAMP for food allergen detection: A strategy with enlarged color variation range and contrast

“…So, certain instruments (such as turbidimeters) or technical expertise are still needed. As for the methods based on the chelating of fluorescent DNA-binding dyes, the result can be easily distinguished by untrained personnel, however, most dyes (such as SYBR Green I) must be added to the solution after amplification with the need of opening the lids ( Girish et al, 2020 , Qian et al, 2017 , Singh et al, 2019 , Zhang et al, 2014c ), greatly increasing the risk of cross-contamination. To this end, visual colorimetric dyes is attractive if it can be pre-added into the LAMP reaction mixtures, without inhibiting the amplification reactions ( Goto et al, 2009 , Li et al, 2016 ).…”