Probe-based real-time PCR method for multilocus melt typing of Xylella fastidiosa strains

• Published in: Journal of microbiological methods Vol. 89; no. 1; pp. 12 - 17
• Main Authors: Brady, Jeff A., Faske, Jennifer B., Ator, Rebecca A., Castañeda-Gill, Jessica M., Mitchell, Forrest L.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.04.2012; Elsevier
• Subjects: Animals; Bacterial plant pathogens; Biological and medical sciences; DNA; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; enzyme-linked immunosorbent assay; epidemiological studies; Fluorophore; Fundamental and applied biological sciences. Psychology; Genotyping; grapes; host plants; Insecta - microbiology; insects; melting; Microbiology; Molecular Sequence Data; Molecular Typing - methods; Oligonucleotide Probes - genetics; Phytopathology. Animal pests. Plant and forest protection; Pierce's disease; Plants - microbiology; quantitative polymerase chain reaction; Quencher; Real-time PCR; Real-Time Polymerase Chain Reaction - methods; Sequence Analysis, DNA; Transition Temperature; Xylella - classification; Xylella - genetics; Xylella - isolation & purification; Xylella fastidiosa; Quencher; Real-time PCR; Genotyping; Fluorophore; Xylella fastidiosa; Pierce's disease; Typing; Plant pathogen; Genotype; Method; Real time; Strain; Polymerase chain reaction
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2012.02.002

Epidemiological studies of Pierce's disease (PD) can be confounded by a lack of taxonomic detail on the bacterial causative agent, Xylella fastidiosa (Xf). PD in grape is caused by strains of Xylella fastidiosa subsp. fastidiosa, but is not caused by other subspecies of Xf that typically colonize plants other than grape. Detection assays using ELISA and qPCR are effective at detecting and quantifying Xf presence or absence, but offer no information on Xf subspecies or strain identity. Surveying insects or host plants for Xf by current ELISA or qPCR methods provides only presence/absence and quantity information for any and all Xf subspecies, potentially leading to false assessments of disease threat. This study uses a series of adjacent-hybridizing DNA melt analysis probes that are capable of efficiently discriminating Xf subspecies and strain relationships in rapid real-time PCR reactions. ► A 1.5hour DNA probe-melt genotyping real-time PCR reaction for Xylella fastidiosa. ► 9 loci genotyped by adjacent-binding DNA probes. ► $0.17 USD of reagents are used per PCR reaction. ► Works on DNA from environmentally-isolated samples (no culturing required). ► Robust even when unexpected genetic variation is encountered.