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TaqMan-based qPCR detection of Xylella fastidiosa subspecies pauca CoDiRO strain

Michael Stulberg: USDA-APHIS-PPQ-S&T-CPHST

<div>The CoDiRO strain of <em>Xylella fastidiosa</em> subsp. <em>pauca</em> is associated with olive quick decline syndrome in southern Italy. Spread of this pathogen to the U.S. could devastate the U.S. olive industry. A TaqMan-based qPCR method identifying this strain would aid U.S. diagnosticians as current identification is limited to more time-consuming methods of sequence analysis and conventional PCR. This subspecies has an open reading frame (ORF) that it shares only with olive-, mulberry-, and hibiscus-infecting <em>X. fastidiosa</em> strains. Detection of this ORF, when combined with a previously designed citrus variegated chlorosis strain-specific assay, allows for CoDiRO differentiation from other <em>pauca</em> strains. We designed qPCR primers and probes (XfOMH) to amplify a target within this ORF. To test our primer set we used a gBlock with the conserved ORF, and two other gene sequences for duplexed qPCR reaction positive control targets (Citrus COX and <em>X. fastidiosa</em> 16S rRNA gene). XfOMH efficiently amplified the gBlock target (104%), and its performance was not significantly changed when duplexed with 16S (100%) or COX (100%) primers. In duplex conditions, XfOMH detected as few as 1.93 LOG gBlock molecules 100% of the time, and 0.23 LOG gBlock molecules 85% of the time. XfOMH did not amplify <em>Xylella</em> from environmental oak samples, but did amplify spiked gBlock targets. This assay will be further developed by testing against a collection of <em>X.</em> <em>fastidiosa</em> strains.</div>