Exploiting the real-time PCR and LAMP for development of a conjunctive new assay (FTP-LAMP) for the specific detection and differentiation of Xylella fastidiosa De Donno and mulberry strains from other subspecies/strains.....

Authors: ELBEAINO, TOUFIC - Mediterranean Agronomic Institute Of Bari; INCERTI, ORNELLO - Mediterranean Agronomic Institute Of Bari; DAKROUB, HIBA - Mediterranean Agronomic Institute Of Bari; VALENTINI, FRANCO - Mediterranean Agronomic Institute Of Bari; Huang, Qi

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/22/2020
Publication Date: 6/23/2020
Citation: Elbeaino, T., Incerti, O., Dakroub, H., Valentini, F., Huang, Q. 2020. Exploiting the real-time PCR and LAMP for development of a conjunctive new assay (FTP-LAMP) for the specific detection and differentiation of Xylella fastidiosa De Donno and mulberry strains from other subspecies/strains...... Journal of Microbiological Methods. https://doi.org/10.1016/j.mimet.2020.105992.
DOI: https://doi.org/10.1016/j.mimet.2020.105992

Interpretive Summary: Xylella fastidiosa (Xf) inhabits plant xylem tissue and causes important diseases in over 30 plant families. Bacterial leaf scorch caused by Xf in mulberry was first reported in 1986, and was found along the east coast of the U.S. In Europe, Xf was reported for the first time in 2013 in Italy, when it was identified in the Apulian olive trees affected by a devastating disease named ‘Olive Quick Decline Syndrome (OQDS)’. ARS Scientists in Beltsville, MD and Italy developed two DNA-based real-time detection methods, TaqMan real-time Polymerase Chain Reaction and Loop-mediated Isothermal Amplification (LAMP), using primers and probe based on a previously -identified sequence that is uniquely present in the Italian olive (De Donno of olive) and American mulberry strains. We also developed a novel assay, referred to as the Fluorescence of TaqMan Probe upon Dequenching - Loop mediated Isothermal Amplification (FTP-LAMP), by combining the LAMP primers and the TaqMan real-time PCR probe, for specific detection of the olive-associated strain in Italy, and differentiation of the De Donno strain from other strains of Xf already reported in Italy and Europe. This technology will be valuable to plant pathologists, entomologists and clinicians interested in diseases caused by Xf.

Technical Abstract: We developed two real-time detection assays, TaqMan real-time PCR and LAMP, using primers and probe designed based on a sequence annotated to code for a Haemagglutinin-related protein (Hg) of Xylella fastidiosa (Xf), a gene uniquely present in the Italian olive (De Donno of olive) and American mulberry strains, for specific detection of the target Xf strains. These assays were validated with DNA samples extracted from Xf-infected plant samples and from two species of insect vectors (Philaenus spumarius, Ps; and Neophilaenus campestris, Nc). Both techniques were proven to be highly sensitive (100 fg of Xf-genomic DNA) and specific to the Italian De Donno and American mulberry strains of Xf. When our LAMP was utilized in a duplex manner by combining with previously published universal primers and probe for detection of all Xf-subspecies and strains, the duplex LAMP showed high versatility in the simultaneous detection and differentiation of the Italian De Donno and American mulberry stains form other subspecies/strains. Furthermore, the Hg gene-specific LAMP primers and TaqMan probe were exploited to develop a new approach; henceforth referred to as the Fluorescence of TaqMan Probe upon Dequenching - Loop mediated Isothermal Amplification (FTP-LAMP). In the FTP-LAMP, the Xf-Hg specific fluorophore-quenched probe was added to a conventional isothermal amplification reaction and fluoresces only when bound to its target, allowing for a sequence-specific detection of the Xf-Italian De Donno and American mulberry strains in a LAMP context. Our FTP-LAMP assay showed to be highly sensitive detecting down to 100 fg genomic DNA of Xf, when tested on Xf-genomic DNA extracted from infected plants, DAS-ELISA-crude saps and insect vectors. Furthermore, the assay showed high specificity (98.7% vs 89% for LAMP) when applied on DNA templates from insect vectors. With the addition of an extra target sequence-specific probe acting as a direct Xf-specific dye, the FTP-LAMP has gained more specificity than the conventional LAMP and reduced one of the main problems of the LAMP assay (false positives) when applied for detection of Xf in insect vectors. To the best of our knowledge, this study reports the development of the first LAMP assay and the first novel FTP-LAMP method for specific detection of the Italian De Donno and the American mulberry strains of Xf. Together with the Xf universal LAMP primers in a multiplexing approach, the FTP-LAMP could represent a useful tool not only for the specific detection of the olive-associated strain in Italy, but also to differentiate the De Donno strain from other strains of Xf already reported in Italy and Europe (Germany, France, Spain and Portugal).