A Loop-Mediated Isothermal Amplification Assay and Sample Preparation Procedure for Sensitive Detection of Xanthomonas fragariae in Strawberry

PloS One
Hehe Wang, W W Turechek

Abstract

Xanthomonas fragariae is a bacterium that causes angular leaf spot of strawberry. Asymptomatic infection is common and contributes to the difficulties in disease management. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay as an efficient method for detection of asymptomatic infections of X. fragariae. In addition, a new method of sample preparation was developed that allows sampling of a larger amount of plant tissue, hence increasing the detection rate in real-life samples. The sample preparation procedure includes an overnight incubation of strawberry tissues in phosphate-buffered saline (PBS), followed by a quick sample concentration and a boiling step to extract DNA for amplification. The detection limit of the LAMP assay was approximately 2×10(3) CFU/mL for pure bacteria culture and 300 CFU/mL for bacteria spiked strawberry leaf and petiole samples. LAMP provided a 2-3 fold lower detection limit than the standard qPCR assay but was faster, and more user-friendly. The LAMP assay should serve as a rapid, sensitive and cost-effective tool for detecting asymptomatic infections of X. fragariae in strawberry nursery stock and contribute to improved disease management.

References

Oct 3, 2006·Journal of Biochemical and Biophysical Methods·Hisatoshi KanekoTatsuo Suzutani
Jan 1, 2007·Structural Equation Modeling : a Multidisciplinary Journal·Stephanie T LanzaJoseph L Schafer
Mar 30, 2012·Medical Decision Making : an International Journal of the Society for Medical Decision Making·Neal V Dawson, Robert Weiss
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Jan 6, 2015·The Journal of Microbiology·Tsugunori NotomiHidetoshi Kanda

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Methods Mentioned

BETA
chemical treatments
enzyme linked immunosorbent assay
PCR
electrophoresis

Software Mentioned

PrimerExplorer
BLAST
SAS
LAMP

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