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Interlaboratory test performance studies for identification of Ralstonia solanacearum and molecular confirmation of its virulence

Tjou-Tam-Sin, Napoleon; Vogelaar, Martijn; Li, XIang; Čermák, Vaclav; Fornefeld, Eva; van der Wolf, Jean Martin; Valentini, Franco; Fraser, Karen; Cara, Magdalena; Yildiz, Nilufer; Yuzbasioglu, Eda; Karahan, Aynur; Ustun, Nursen; Kreuze, Jan


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    <subfield code="a">&lt;p&gt;The EU directive 2006/63/EC describes a detailed protocol for the official testing of &lt;em&gt;Ralstonia solanacearum&lt;/em&gt;, that is internationally recognized and has been implemented in many diagnostic laboratories across Europe and beyond. In this protocol, the confirmation of the identity of the bacterium is performed by a laborious, time-consuming and expensive pathogenicity test. Additionally, the described protocols involved in the official testing of &lt;em&gt;Ralstonia solanacearum&lt;/em&gt; (for detection and/or identification) require a lot of time, a high level of quarantine measures, and a high degree of expertise. The aim of this project was to develop, and evaluate molecular diagnostic methods for the detection and identification of &lt;em&gt;Ralstonia solanacearum&lt;/em&gt; and verification of its virulence that would be faster, more specific and robust. Based on the current taxonomic insights, the high degree of heterogenity present inside the &lt;em&gt;Ralstonia solanacearum&lt;/em&gt; species complex (RSSC) has resulted in the clear distinction of three new species inside this complex; the requirement defined in this Euphresco project was that the new molecular tests could be optimally implemented for the detection and identification of &lt;em&gt;Ralstonia solanacearum&lt;/em&gt; and &lt;em&gt;Ralstonia pseudosolanacearu&lt;/em&gt;m, but not of &lt;em&gt;Ralstonia syzygii&lt;/em&gt;.&lt;/p&gt;

&lt;p&gt;The two main objectives of the project were:&lt;/p&gt;

&lt;p&gt;1) To identify &lt;em&gt;Ralstonia solanacearum &lt;/em&gt;virulence genes, and subsequently develop a PCR test on those virulence genes. The development of such a test could substitute the pathogenicity test required by EU directive 2006/63/EC for complete diagnosis of &lt;em&gt;Ralstonia solanacearum&lt;/em&gt;.&lt;/p&gt;

&lt;p&gt;2) To verify other molecular methods to detect or identify &lt;em&gt;Ralstonia solanacearum &lt;/em&gt;strains. These methods include the real-time Loop-mediated isothermal amplication test (LAMP) and the recombinase-polymerase amplification assay (RPA).&lt;/p&gt;</subfield>
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