Specific detection of Ralstonia solanacearum 16S rRNA sequences by AmpliDet RNA

Abstract

The potential of AmpliDet RNA for specific detection of Ralstonia solanacearum in potato tuber samples and surface water was demonstrated. AmpliDet RNA is a procedure based on nucleic acid sequence based amplification (NASBA) of RNA sequences and homogeneous real time detection of NASBA amplicons with a molecular beacon. The procedure is carried out in sealed tubes, thus reducing the risks for carry-over contamination. AmpliDet RNA enabled reliable detection of specific 16S rRNA sequences of R. solanacearum in total RNA extracts from potato tuber samples in 90 min at a level of 10 cells per reaction, equivalent to ca. 104 cells ml-1 of sample. In surface water, AmpliDet RNA allowed detection of R. solanacearum at a level of 10 cfu ml-1, after concentrating bacteria from 200 ml of surface water into 1 ml of surface water by centrifugation. All strains of R. solanacearum and a strain of R. syzygii were positive in AmpliDet RNA, but not other (related) bacterial species. Ralstonia solanacearum (race 3, biovar 2) could be detected reliably in 18 naturally infected potato tuber samples containing varying concentrations of cells. Ninety-one negative tuber samples, from which no R. solanacearum was isolated, were tested in AmpliDet RNA, including 23 samples containing bacteria (cross-) reacting with antibodies against R. solanacearum in immunofluorescence (IF) cell-staining. Only one negative sample, containing high numbers of IF-positive cells, was positive in AmpliDet RNA