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

Lindsey D Thiessen,Walter F Mahaffee,Tara M Neill

doi:10.7717/peerj.4639

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 Erysiphe necator 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.

Highlights

Molecular techniques, such as PCR, are capable of being used to detect specific pathogens in air samples with high sensitivity and specificity (Carisse, Bacon & Lefebvre, 2009; Carisse et al, 2009b; Falacy et al, 2007; Thiessen et al, 2016; West et al, 2008)
The L-quantitative LAMP (qLAMP) spore quantification was significantly lower than the quantitative PCR (qPCR) quantification when daily samples were collected in 2013 (P < 0.001) (Fig. 3A), but the biweekly Lab-conducted qLAMP (L-qLAMP) and qPCR sample quantification was not significantly different in 2013 (P = 0.14) (Fig. 3B)
The qLAMP assay consistently underrepresented spore quantities later in the growing season compared to the qPCR assay, which may be due to an increase in the presence of PCR inhibitors found in air samples (Wilson, 1997) that may not have been removed by the rapid Chelex DNA extraction

Summary

Introduction

Molecular techniques, such as PCR, are capable of being used to detect specific pathogens in air samples with high sensitivity and specificity (Carisse, Bacon & Lefebvre, 2009; Carisse et al, 2009b; Falacy et al, 2007; Thiessen et al, 2016; West et al, 2008). LAMP can use relatively inexpensive and mobile equipment and utilizes the Bst polymerase that has a high tolerance to reaction inhibitors (Kubota et al, 2011), which allows for quick, minimal DNA extraction protocols. These traits make LAMP useful in field detection assays (Harper, Ward & Clover, 2010; Kubota et al, 2008; Temple & Johnson, 2011; Tomlinson, Barker & Boonham, 2007; Tomlinson, Dickinson & Boonham, 2010)

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Conclusion