REAL TIME PCR FOR CLINICAL DETECTION AND DIFFERENTIATION OF XYLELLA FASTIDOSA STRAINS

• Author(s): Civerolo, Ed; Francis, Marta; Lin, Hong;
• Abstract: The overall goal of this work is to develop reliable protocols for the clinical detection and identification of Xylella fastidiosa (Xf) strains. The objectives are to (1) apply PCR-based methods to detect low populations of Xf strains causing the Pierces disease of grapevines infected grape tissue; and (2) to distinguish different Xf strains in naturally-occurring single or mixed infections in different hosts, as well as in insect vectors. A major problem is the presence of PCR inhibitors in the grape tissue extracts that result in false negative results. A simplified method for the isolation of grape tissue DNA, using a single tube for grinding and extraction, was developed. Two real time PCR systems were developed for the generic detection of Xf strains and the specific detection of the Xf-PD strain or pathotype based on the currently available genomic sequences of four Xf strains. One system, based on a set of primers, designated HL5/HL6, and a probe labeled with FAM (HL5/HL6-FAM) as a fluorescent dye, detected four Xf strains (PD, almond leaf scorch (ALS), oleander leaf scorch (OLS)) and citrus variegated chlorosis (CVC) DNA. The specificity of the primers was tested against several other plant pathogenic bacteria and endophytic bacteria isolated from grape, no amplification products were obtained using 103-104 cells/reaction. The Xf-specific amplification product was 221 bp. As few as 5 bacteria per reaction were detected using this system. Standard curves were obtained with intact bacteria in water and in preparations containing grape leaf petiole DNA from the equivalent of 1 mg of fresh grape tissue per reaction. The Ct values ranged from 20 cycles for 105 bacteria per reaction to 36 cycles for 5 bacteria per reaction (r2 0.9). A second system, based on a set of Xf-PD specific primers, designated HL7/HL8, and a probe labeled with TET (HL7/HL8-TET) as a fluorescent dye, was developed. The Xf-specific product in this case was 302bp. This set of primers specifically distinguished Xf-PD from Xf-ALS and Xf-OLS, as well as from Xf-CVC DNA. The use of these two systems permits the detection of the Xf strains and the specific detection of Xf-PD, and could be used to detect as few as 5 bacteria per reaction.
• Publication Date: Aug 2003
• Journal: 2003 Pierce's Disease Research Symposium