| Popis: |
Surveys for Xylella fastidiosa are now mandatory in the EU Member States and genetically diverse strains, i.e. those capable of infecting different hosts, have been intercepted in the currently known EU outbreaks and/or containment areas. Given the long list of the EU susceptible host plants, currently comprising more than 50 plant species, there is an compelling need to develop robust and rapid diagnostic tests (ready-to-use and on-site tests) suitable for testing and screening different plant matrices and large numbers of samples. To this end, we have compared membrane-capture-based methods and ready-to-use kits, the latter based on isothermal nucleic acid amplification techniques. Indeed, a chloroform-free method based on the use of the Maxwell® RSC PureFood GMO and Authentication Kit (Promega) was evaluated on different plant matrices, as an alternative protocol for recovering total DNA suitable for identifying X. fastidiosa in real-time PCR (qPCR) reactions, so far the most widely used diagnostic method. In a first validation test, 90 olive field-trees were tested simultaneously using different procedures: FTA-ELISA, DTBIA, qPCR assay on membrane-captured DNA, loop-mediated isothermal amplification/LAMP on fresh sap or on stored tissue-imprinted membranes, and recombinase polymerase amplification/RPA.Performance criteria for each test were determined by comparing the results with those obtained in qPCR assays performed according to Harper et al. (2010). The results, while suggesting that different types of membranes can be used for capturing bacterial cells for a subsequent serological or molecular detection, showed that all these approaches produced lower accuracy values than LAMP, RPA and standard qPCR. The chloroform-free DNA purification kit herein tested made it possible to recover DNA templates of high quality with standardised yields, suitable for the detection of the bacterium using the qPCR protocols currently validated for X. fastidiosa. |
