Submitted to: CDFA Pierce's Disease Control Program Research Symposium
Publication Type: Proceedings
Publication Acceptance Date: 11/27/2006
Publication Date: 11/27/2006
Citation: Chen, J., Livingston, S., Civerolo, E.L., Kirkpatrick, B. 2006. Towards the development of a sequence-based pcr system for detection and genomic study of xylella fastidosa strains important to california. In: Proceedings of the CDFA Pierce's Disease Control Program Research Symposium, November 27-29, 2006, San Diego, CA. p. 127-131. Interpretive Summary: Primer selection and sample preparation to provide template DNA are two key components for the success of polymerase chain reaction (PCR). Most of the currently tested PCR primers for Xylella fastidiosa detection target conserved genes and discriminate X. fastidiosa pathotypes from grape and almond. This study identified diversity within the same pathotype in a highly variable region called pspB. Further study on this gene will facilitate understanding of bacterial pathogenicity and environmental adaptation. We also evaluated a PCR procedure with minimal sample preparation: pulverized freeze-dried tissue PCR (PFT-PCR). The results indicated that PFT-PCR had a high predictive value (90.8%) for positive results, but a low predictive value for negative results (29.7%). We recommend the PFT-PCR for high throughput detection of X. fastidiosa.
Technical Abstract: We report two of our recent efforts for improvement of PCR detection and genomic analysis of Xylella fastidiosa. We evaluated the use of PCR primers from a hyper-variable locus to monitor diversity within strains of the same pathotype. The pspB (PD1208) locus, encoding a serine protease, was selected and analyzed. It was observed that tandem repeat sequences in pspB locus were highly variable from strain to strain. The biological significance of this hyper-variation is unknown. We also evaluated a simple sample preparation method for template DNA. The pulverized freeze-dried tissue PCR (PFT-PCR) test was compared to the “gold standard” pathogen isolation method. Our results indicated that PFT-PCR had a high predictive value (90.8%) for true positive samples, but a low predictive value for true negative results (29.7%), indicating that a PFT-PCR result is best suited to confirm the presence of X. fastidiosa in a sample.