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Design and performance of a 16S rRNA-targeted oligonucleotide probe for detection of members of the genus Bdellovibrio by fluorescence in situ hybridization.

ABSTRACT: A 16S rRNA-targeted, Cy3-labeled oligonucleotide probe was designed to detect members of the genus Bdellovibrio by fluorescence in situ hybridization. Specific hybridization conditions were established; however, the detection of bdellovibrios in environmental samples required enrichment, confirming that Bdellovibrio spp. are not present in large numbers in the environment.

SUBMITTER: Mahmoud KK

PROVIDER: S-EPMC2168191 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

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Design and performance of a 16S rRNA-targeted oligonucleotide probe for detection of members of the genus Bdellovibrio by fluorescence in situ hybridization.

Mahmoud Khaled K KK McNeely Damian D Elwood Chelsea C Koval Susan F SF

Applied and environmental microbiology 20070928 22

A 16S rRNA-targeted, Cy3-labeled oligonucleotide probe was designed to detect members of the genus Bdellovibrio by fluorescence in situ hybridization. Specific hybridization conditions were established; however, the detection of bdellovibrios in environmental samples required enrichment, confirming that Bdellovibrio spp. are not present in large numbers in the environment. ...[more]

PMID: 17905886

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Project description:Prorocentrum donghaiense is a common but dominant harmful algal bloom (HAB) species, which is widely distributed along the China Sea coast. Development of methods for rapid and precise identification and quantification is prerequisite for early-stage warning and monitoring of blooms due to P. donghaiense. In this study, sequences representing the partial large subunit rDNA (D1-D2), small subunit rDNA and internal transcribed spacer region (ITS-1, 5.8S rDNA and ITS-2) of P. donghaiense were firstly obtained, and then seven candidate DNA probes were designed for performing fluorescence in situ hybridization (FISH) tests on P. donghaiense. Based on the fluorescent intensity of P. donghaiense cells labeled by the DNA probes, the probe DP0443A displayed the best hybridization performance. Therefore, a PNA probe (PP0443A) analogous to DP0443A was used in the further study. The cells labeled with the PNA probe displayed more intensive green fluorescence than that labeled with its DNA analog. The PNA probe was used to hybridize with thirteen microalgae belonging to five families, i.e., Dinophyceae, Prymnesiophyceae, Raphidophyceae, Chlorophyceae and Bacillariophyceae, and showed no visible cross-reaction. Finally, FISH with the probes PP0443A and DP0443A and light microscopy (LM) analysis aiming at enumerating P. donghaiense cells were performed on the field samples. Statistical comparisons of the cell densities (cells/L) of P. donghaiense in the natural samples determined by FISH and LM were performed using one-way ANOVA and Duncan's multiple comparisons of the means. The P. donghaiense cell densities determined by LM and the PNA probe are remarkably higher than (p<0.05) that determined by the DNA probe, while no significant difference is observed between LM and the PNA probe. All results suggest that the PNA probe is more sensitive that its DNA analog, and therefore is promising for the monitoring of harmful algal blooms of P. donghaiense in the future.

Dataset Information

Design and performance of a 16S rRNA-targeted oligonucleotide probe for detection of members of the genus Bdellovibrio by fluorescence in situ hybridization.

Publications

Design and performance of a 16S rRNA-targeted oligonucleotide probe for detection of members of the genus Bdellovibrio by fluorescence in situ hybridization.

Similar Datasets

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