Non-instrumented DNA isolation, amplification and microarray-based hybridization for a rapid on-site detection of devastating Phytophthora kernoviae
| Autor: | Jürgen Popp, Karina Weber, Lydia Schwenkbier, Sebastian Sailer, Anne Rudloff, Dana Cialla-May, Sibyll Pollok |
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| Rok vydání: | 2015 |
| Předmět: |
Phytophthora
Time Factors biology Base Sequence Chemistry DNA–DNA hybridization Hybridization probe Nucleic Acid Hybridization DNA Amplicon biology.organism_classification Biochemistry DNA extraction Molecular biology Analytical Chemistry Enzyme binding Nucleic acid thermodynamics Phytophthora kernoviae Electrochemistry Environmental Chemistry DNA Probes Spectroscopy Oligonucleotide Array Sequence Analysis |
| Zdroj: | The Analyst. 140(19) |
| ISSN: | 1364-5528 |
| Popis: | A rapid and simple instrument-free detection system was developed for the identification of the plant pathogen Phytophthora kernoviae (P. kernoviae). The on-site operable analysis steps include magnetic particle based DNA isolation, helicase-dependent amplification (HDA) and chip-based DNA hybridization. The isothermal approach enabled the convenient amplification of the yeast GTP-binding protein (Ypt1) target gene in a miniaturized HDA-zeolite-heater (HZH) by an exothermic reaction. The amplicon detection on the chip was performed under room temperature conditions – either by successive hybridization and enzyme binding or by a combined step. A positive signal is displayed by enzymatically generated silver nanoparticle deposits, which serve as robust endpoint signals allowing an immediate visual readout. The hybridization assay enabled the reliable detection of 10 pg μL(-1) target DNA. This is the first report of an entirely electricity-free, field applicable detection approach for devastating Phytophthora species, exemplarily shown for P. kernoviae. |
| Databáze: | OpenAIRE |
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