Optimization of loop-mediated isothermal amplification assay for sunflower mildew disease detection.
Plasmopara halstedii
Loop-mediated isothermal amplification (LAMP)
Plant disease detection
Plant pathogens
Sunflower downy mildew
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
05 Oct 2024
05 Oct 2024
Historique:
received:
23
05
2024
accepted:
04
09
2024
medline:
6
10
2024
pubmed:
6
10
2024
entrez:
5
10
2024
Statut:
epublish
Résumé
Loop-Mediated Isothermal Amplification (LAMP) represents a valuable technique for DNA/RNA detection, known for its exceptional sensitivity, specificity, speed, accuracy, and affordability. This study focused on optimizing a LAMP-based method to detect early signs of Plasmopara halstedii, the casual pathogen of sunflower downy mildew, a severe threat to sunflower crops. Specifically, a set of six LAMP primers (two outer, two inner, and two loop) were designed from P. halstedii genomic DNA, targeting the ribosomal Large Subunit (LSU). These primers were verified by in silico analysis and experimental validation using both target and non-target species' DNAs. Optimizations encompassing reaction conditions (temperature, time) and component concentrations (magnesium, Bst DNA polymerase, primers, and dNTP) were determined. Validation of these optimizations was performed by agarose gel electrophoresis. Furthermore, various colorimetric chemicals (Neutral Red, Hydroxynaphthol Blue, SYBR Safe, Thiazole Green) were evaluated to facilitate method analysis, and the real-time analysis has been optimized, presenting multiple approaches for detecting sunflower downy mildew using the LAMP technique. The analytical sensitivity of the method was confirmed by detecting P. halstedii DNA concentrations as low as 0.5 pg/μl. This pioneering study, establishing P. halstedii detection through the LAMP method, stands as unique in its field. The precision, robustness, and practicality of the LAMP protocol make it an ideal choice for studies focusing on sunflower mildew, emphasizing its recommended use due to its operational ease and reliability.
Identifiants
pubmed: 39369029
doi: 10.1038/s41598-024-72228-y
pii: 10.1038/s41598-024-72228-y
doi:
Substances chimiques
DNA Primers
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
23224Subventions
Organisme : The Scientific and Technological Research Council of Türkiye
ID : 2210A
Organisme : The Research Fund of Istanbul University
ID : FYL 2022-39343
Informations de copyright
© 2024. The Author(s).
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