Dataset Information

Ca2+ in Hybridization Solutions for Fluorescence in situ Hybridization Facilitates the Detection of Enterobacteriaceae.

ABSTRACT: Fluorescence in situ hybridization (FISH) has been employed to identify microorganisms at the single cell level under a microscope. Extensive efforts have been made to improve and extend the FISH technique; however, the development of a widely applicable protocol is a continuing challenge. The present study evaluated the effects of divalent cations in the hybridization solution on the FISH-based detection of various species of bacteria and archaea with rRNA-targeted probes. A flow cytometric analysis after FISH with a standard hybridization buffer detected positive signals from less than 30% of Escherichia coli IAM 1264 cells. However, the number of cells with positive signals increased to more than 90% after the addition of calcium chloride to the hybridization buffer. Mn2+ also had positive effects, whereas Mg2+ did not. The positive effects of Ca2+ were similarly observed for bacteria belonging to Enterobacteriaceae, including Enterobacter sakazakii IAM 12660T, E. aerogenes IAM 12348, Klebsiella planticola IAM 14202, and Salmonella enterica subsp. enterica serovar Typhimurium strain LT2. These results indicate that the supplementation of Ca2+ to the hybridization buffer for FISH contributes to the efficient detection of Enterobacteriaceae cells.

SUBMITTER: Haruta S

PROVIDER: S-EPMC5478537 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Ca<sup>2+</sup> in Hybridization Solutions for Fluorescence in situ Hybridization Facilitates the Detection of Enterobacteriaceae.

Haruta Shin S Iino Takao T Ohkuma Moriya M Suzuki Ken-Ichiro KI Igarashi Yasuo Y

Microbes and environments 20170518 2

Fluorescence in situ hybridization (FISH) has been employed to identify microorganisms at the single cell level under a microscope. Extensive efforts have been made to improve and extend the FISH technique; however, the development of a widely applicable protocol is a continuing challenge. The present study evaluated the effects of divalent cations in the hybridization solution on the FISH-based detection of various species of bacteria and archaea with rRNA-targeted probes. A flow cytometric ana ...[more]

PMID: 28515389

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Project description:BACKGROUND: Dengue fever (DF) and dengue hemorrhagic fever (DHF) represent a global challenge in public health. It is estimated that 50 to 100 million infections occur each year causing approximately 20,000 deaths that are usually linked to severe cases like DHF and dengue shock syndrome. The causative agent of DF is dengue virus (genus Flavivirus) that comprises four distinct serotypes (DENV-1 to DENV-4). Fluorescence in situ hybridization (FISH) has been used successfully to detect pathogenic agents, but has not been implemented in detecting DENV. To improve our understanding of DENV infection and dissemination in host tissues, we designed specific probes to detect DENV in FISH assays. METHODS: Oligonucleotide probes were designed to hybridize with RNA from the broadest range of DENV isolates belonging to the four serotypes, but not to the closest Flavivirus genomes. Three probes that fit the criteria defined for FISH experiments were selected, targeting both coding and non-coding regions of the DENV genome. These probes were tested in FISH assays against the dengue vector Aedes albopictus (Diptera: Culicidae). The FISH experiments were led in vitro using the C6/36 cell line, and in vivo against dissected salivary glands, with epifluorescence and confocal microscopy. RESULTS: The three 60-nt oligonucleotides probes DENV-Probe A, B and C cover a broad range of DENV isolates from the four serotypes. When the three probes were used together, specific fluorescent signals were observed in C6/36 infected with each DENV serotypes. No signal was detected in either cells infected with close Flavivirus members West Nile virus or yellow fever virus. The same protocol was used on salivary glands of Ae. albopictus fed with a DENV-2 infectious blood-meal which showed positive signals in the lateral lobes of infected samples, with no significant signal in uninfected mosquitoes. CONCLUSION: Based on the FISH technique, we propose a way to design and use oligonucleotide probes to detect arboviruses. Results showed that this method was successfully implemented to specifically detect DENV in a mosquito cell line, as well as in mosquito salivary glands for the DENV-2 serotype. In addition, we emphasize that FISH could be an alternative method to detect arboviruses in host tissues, also offering to circumvent the discontinuity of antibodies used in immunofluorescent assays.

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

Ca2+ in Hybridization Solutions for Fluorescence in situ Hybridization Facilitates the Detection of Enterobacteriaceae.

Publications

Ca<sup>2+</sup> in Hybridization Solutions for Fluorescence in situ Hybridization Facilitates the Detection of Enterobacteriaceae.

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