Development of a genome-informed loop-mediated isothermal amplification assay for rapid and specific detection of Xanthomonas euvesicatoria

Adriana Larrea-Sarmiento,Gamze Boluk,Jeff Jones,Lilly Fatdal,Anne Alvarez,Upasana Dhakal,Mathews Paret,Mohammad Arif,Daniel Jenkins,Amanda Strayer-Scherer

doi:10.1038/s41598-018-32295-4

Abstract

Bacterial spot (BS), caused by Xanthomonas euvesicatoria, X. vesicatoria, X. gardneri and X. perforans, is an economically important bacterial disease of tomato and pepper. Symptoms produced by all four species are nearly indistinguishable. At present, no point-of-care diagnostics exist for BS. In this research, we examined genomes of X. euvesicatoria, X. vesicatoria, X. gardneri, X. perforans and other species of Xanthomonas; the unique gene recG was chosen to design primers to develop a loop-mediated isothermal amplification (LAMP) assay to rapidly and accurately identify and differentiate X. euvesicatoria from other BS causing Xanthomonas sp. using a field-deployable portable BioRangerTM instrument. Specificity of the developed assay was tested against 39 strains of X. euvesicatoria and 41 strains of other species in inclusivity and exclusivity panels, respectively. The assay detection limit was 100 fg (~18 genome copies) of genomic DNA and 1,000 fg in samples spiked with tomato DNA. The assay unambiguously detected X. euvesicatoria in infected tomato plant samples. Concordant results were obtained when multiple operators performed the test independently. No false positives and false negatives were detected. The developed LAMP assay has numerous applications in diagnostics, biosecurity and disease management.

Highlights

Bacterial spot (BS) of tomato (Solanum lycopersicum) and pepper (Capsicum spp.) is one of the most serious and economically important diseases worldwide
Regardless of causing similar symptoms on the same hosts, X. euvesicatoria, X. vesicatoria, X. gardneri and X. perforans were clustered in two subgroups (Fig. 1B)
We developed and validated a BioRangerTM and colorimetric based loop-mediated isothermal amplification (LAMP) protocol for specific, sensitive, reliable and robust detection and differentiation of X. euvesicatoria, a causal agent of bacterial spot disease affecting both tomato and pepper

Summary

Introduction

Bacterial spot (BS) of tomato (Solanum lycopersicum) and pepper (Capsicum spp.) is one of the most serious and economically important diseases worldwide. The disease is caused by four species of Xanthomonas, Xanthomonas euvesicatoria, X. perforans, X. vesicatoria and X. gardneri[1]. Xanthomonads are identified using Multilocus Sequence Typing (MLST), Amplified Fragment Length Polymorphism (AFLP), DNA-DNA hybridization, and other polymerase chain reaction-based methods including end-point PCR, multiplex PCR, and real-time quantitative PCR (qPCR)[10,11,12]. LAMP is the most popular and widely used isothermal-based detection method because its rapid, ease to perform and has greater sensitivity and is compatible with numerous detection chemistries. Most importantly, it can be performed at the point-of-care. The visualization of the amplification products can be obtained using several methods including gel electrophoresis, measuring turbidity and visually evaluating the color change by SYBR Green stain

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Conclusion