New lateral flow immunoassay for on-site detection of Erwinia amylovora and its application on various organs of infected plants

Razo, Shyatesa C.; Safenkova, Irina V.; Drenova, Natalia V.; Kharchenko, A. A.; Tsymbal, Y.S.; Varitsev, Yuri A.; Zherdev, Anatoly V.; Pakina, Elena N.; Dzantiev, Boris B.

doi:10.1016/j.pmpp.2021.101637

Cited by 7 publications

(2 citation statements)

References 17 publications

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“…However, in the article comparing LAMP and PCR [ 52 ], LAMP-based primers from [ 53 ] and qPCR assays showed high specificity for E. amylovora and were able to detect up to ×10 3 CFU/mL. Regarding immunoassays [ 41 , 51 ], all test systems based on isothermal amplifications turned out to be expectedly more sensitive, by 2–4 orders of magnitude depending on the type of test system.…”

Section: Resultsmentioning

confidence: 99%

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Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora

et al. 2022

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Isothermal amplifications allow for the highly sensitive detection of nucleic acids, bypassing the use of instrumental thermal cycling. This work aimed to carry out an experimental comparison of the four most promising techniques: recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) coupled with lateral flow test or coupled with additional amplification based on CRISPR/Cas12a resulting from the fluorescence of the Cas12a-cleaved probe. To compare the four amplification techniques, we chose the bacterial phytopathogen Erwinia amylovora (causative agent of fire blight), which has a quarantine significance in many countries and possesses a serious threat to agriculture. Three genes were chosen as the targets and primers were selected for each one (two for RPA and six for LAMP). They were functionalized by labels (biotin, fluorescein) at the 5′ ends for amplicons recognition by LFT. As a result, we developed LAMP-LFT, LAMP-CRISPR/Cas, RPA-LFT, and RPA-CRISPR/Cas for E. amylovora detection. The detection limit was 104 CFU/mL for LAMP-LFT, 103 CFU/mL for LAMP-CRISPR/Cas, and 102 CFU/mL for RPA-LFT and RPA-CRISPR/Cas. The results of four developed test systems were verified by qPCR on a panel of real samples. The developed assays based on RPA, LAMP, CRISPR/Cas12a, and LFT are rapid (30–55 min), user-friendly, and highly sensitive for E. amylovora detection. All proposed detection methods can be applied to fire blight diagnosis and effective management of this disease.

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Section: Resultsmentioning

confidence: 99%

“…For measurement of optical density (OD), NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA) was used. A concentration of about 2 × 10 9 colony-forming unit (CFU)/mL was estimated at 0.1 OD, at a wavelength of 600 nm [ 41 ].…”

Section: Methodsmentioning

confidence: 99%

Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora

et al. 2022

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Erwinia

Coutinho,

Shin

2023

Bergey's Manual of Systematics of Archaea and Bacteria

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er.win'i.a. N.L. fem. n. Erwinia , named after Erwin F. Smith. Proteobacteria / Gammaproteobacteria / Enterobacterales / Erwiniaceae / Erwinia Cells of the genus Erwinia are Gram‐stain‐negative rods, 0.5–1.0 × 1.0–3.0 μm, and occur alone or in pairs, sometimes in chains. Cells are motile by means of peritrichous flagella. They do not produce indole. They are mostly facultatively anaerobic, catalase‐positive, and oxidase‐ and urease‐negative. They utilize d ‐arabinose, fructose, fumarate, galactose, gluconate, glucose, glycerol, malate, β‐methyl‐glucoside, N‐ acetyl‐ d ‐glucosamine, ribose, trehalose, and succinate but not benzoate, butanol, l ‐fucose, glycogen, methanol, oxalate, propionate, or sorbose as the sole carbon sources. The major fatty acids are C 12:0 , C 14:0 , and C 16:0 . The known habitats are plants, where they cause vascular wilts and soft rots. They are also part of the epiphytic flora. They have also been isolated from insects and in one case from human skin. DNA G + C content (mol%) : 51.1–56.4%. Type species : Erwinia amylovora Winslow et al. 1920 AL (basonym: Micrococcus amylovorus Burrill 1882).

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