Development of LAMP and real-time PCR methods for the rapid detection of Xylella fastidiosa for quarantine and field applications

Abstract

Xylella fastidiosa is a regulated plant pathogen in many parts of the world. To increase diagnostic capability of X. fastidiosa in the field, a loop-mediated isothermal amplification (LAMP) and real-time polymerase chain reaction (PCR) assay were developed to the rimM gene of X. fastidiosa and evaluated for specificity and sensitivity. Both assays were more robust than existing published assays for detection of X. fastidiosa when screened against 20 isolates representing the four major subgroups of the bacterium from a range of host species. No cross-reaction was observed with DNA from healthy hosts or other bacterial species. The LAMP and real-time assays could detect 250 and 10 copies of the rimM gene, respectively, and real-time sensitivity was comparable with an existing published real-time PCR assay. Hydroxynapthol blue was evaluated as an endpoint detection method for LAMP. When at least 500 copies of target template were present, there was a noticeable color change indicating the presence of the bacterium. Techniques suitable for DNA extraction from plant tissue in situ were compared with a standard silica-column-based laboratory extraction method. A portable PickPen and magnetic bead system could be used to successfully extract DNA from infected tissue and could be used in conjunction with LAMP in the field.