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Development of a quantitative loop-mediated isothermal amplification assay for the field detection of . | LitMetric

Development of a quantitative loop-mediated isothermal amplification assay for the field detection of .

PeerJ

Agricultural Research Service, United States Department of Agriculture, Corvallis, OR, USA.

Published: April 2018

AI Article Synopsis

  • Plant pathogen detection systems, like LAMP assays, help manage grape powdery mildew but have issues with false results due to low DNA detection.
  • A new quantitative LAMP (qLAMP) assay was tested by grape growers in Oregon using handheld devices and tablets, but results showed unreliable inoculum quantification over time.
  • Despite initial sensitivity, the qLAMP assay's effectiveness diminished during testing, making it less suitable compared to other molecular tools for managing grape powdery mildew.

Article Abstract

Plant pathogen detection systems have been useful tools to monitor inoculum presence and initiate management schedules. More recently, a loop-mediated isothermal amplification (LAMP) assay was successfully designed for field use in the grape powdery mildew pathosystem; however, false negatives or false positives were prevalent in grower-conducted assays due to the difficulty in perceiving the magnesium pyrophosphate precipitate at low DNA concentrations. A quantitative LAMP (qLAMP) assay using a fluorescence resonance energy transfer-based probe was assessed by grape growers in the Willamette Valley of Oregon. Custom impaction spore samplers were placed at a research vineyard and six commercial vineyard locations, and were tested bi-weekly by the lab and by growers. Grower-conducted qLAMP assays used a beta-version of the Smart-DART handheld LAMP reaction devices (Diagenetix, Inc., Honolulu, HI, USA), connected to Android 4.4 enabled, Bluetooth-capable Nexus 7 tablets for output. Quantification by a quantitative PCR assay was assumed correct to compare the lab and grower qLAMP assay quantification. Growers were able to conduct and interpret qLAMP results; however, the inoculum quantification was unreliable using the beta-Smart-DART devices. The qLAMP assay developed was sensitive to one spore in early testing of the assay, but decreased to >20 spores by the end of the trial. The qLAMP assay is not likely a suitable management tool for grape powdery mildew due to losses in sensitivity and decreasing costs and portability for other, more reliable molecular tools.

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912203PMC
http://dx.doi.org/10.7717/peerj.4639DOI Listing

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