title: A new and sensitive Co-operational polymerase chain reaction for rapid detection of Ralstonia solanacearum in water.
abstract: Three primers from 16S rRNA were successfully assayed simultaneously in one reaction for sensitive detection of Ralstonia solanacearum in watercourses. The protocol is a modification of the Co-operational polymerase chain reaction (Co-PCR), which allows the simultaneous and co-operational action of the primers. It specifically amplified R. solanacearum strains belonging to biovars 1, 2 and 4. No products were obtained from any of the 162 unidentified isolates from river water. The sensitivity of the assay was <1 cfu/ml as determined by analysis of heat-treated water samples spiked with R. solanacearum, also containing indigenous microbiota up to 10(5) cfu/ml. The developed Co-PCR assay was more sensitive than other standard PCR assays in the analysis of 51 Spanish environmental water samples. Namely 31.3% of the samples were positive using the newly developed assay, whereas 13.7% or less positive samples were found with the other protocols. The Co-PCR improves the detection sensitivity of R. solanacearum and provides an important tool for its routine detection from environmental water samples and for epidemiological studies.
mesh_terms: D001483:Base Sequence; D003124:Colorimetry; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D043368:Ralstonia solanacearum / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014871:Water Microbiology