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Soaking petiole cross-sections provides an alternative method to prepare samples for Xylella fastidiosa detection using the AmplifyRP kit

Rugang Li: Agdia, Inc.

<div><em>Xylella fastidiosa</em> (<em>Xf</em>), a xylem-limited bacterium, infects more than 350 plant species (including hybrids) from 204 genera of 75 different botanical families. Establishing optimized DNA extraction protocols is important for achieving reliable detection of <em>Xf</em> from different plant species. Traditional DNA extraction by grinding plant tissues and subsequent purification steps is time-consuming and laborious. Agdia has developed a sensitive isothermal AmplifyRP kit for detecting <em>Xf</em> using crude extract. However, the positive response can be inhibited when fully macerated crude extracts (1:10 wt/vol) from select hosts and/or tissue types are used directly in the reaction. Here we have developed a simple method, called Soaking Petiole Cross-Sections (SPCS), to release <em>Xf</em> DNA from petiole tissue in AMP1 buffer (Agdia). Petiole cross-sections (2-3 mm in thickness) were prepared from <em>Xf</em> infected-almond, blueberry, grapevine, and maple and soaked in AMP1 buffer (1:10 wt/vol) for 10 min, 30 min, and 5 hr at room temperature. No inhibition was observed in serial dilutions of the soaking extract for the above tested plant species. Soaking for 10 min was enough to generate a similar amplification signal as for 5 hr. For leaf tissues that contain lower bacterial concentrations and/or more inhibitors SPCS can be used as an alternative method for <em>Xf</em> detection using the AmplifyRP kit. Extensive comparative evaluation of other host species is ongoing.</div>