@article{fdi:010070083, title = {{S}pecificity re-evaluation of oligonucleotide probes for the detection of marine picoplankton by tyramide signal amplification-fluorescent in situ hybridization}, author = {{R}iou, {V}. and {P}eriot, {M}. and {B}iegala, {I}sabelle}, editor = {}, language = {{ENG}}, abstract = {{O}ligonucleotide probes are increasingly being used to characterize natural microbial assemblages by {T}yramide {S}ignal {A}mplification-{F}luorescent in situ {H}ybridization ({TSA}-{FISH}, or {CA}talysed {R}eporter {D}eposition {CARD}-{FISH}). {I}n view of the fast-growing r{RNA} databases, we re-evaluated the in silico specificity of eleven bacterial and eukaryotic probes and competitor frequently used for the quantification of marine picoplankton. {W}e performed tests on cell cultures to decrease the risk for non-specific hybridization, before they are used on environmental samples. {T}he probes were confronted to recent databases and hybridization conditions were tested against target strains matching perfectly with the probes, and against the closest non-target strains presenting one to four mismatches. {W}e increased the hybridization stringency from 55 to 65% formamide for the {E}ub338+{E}ub{II}+{E}ub{III} probe mix to be specific to the {E}ubacteria domain. {I}n addition, we found that recent changes in the {G}ammaproteobacteria classification decreased the specificity of {G}am42a probe, and that the {R}oseo536{R} and {R}os537 probes were not specific to, and missed part of the {R}oseobacter clade. {C}hanges in stringency conditions were important for bacterial probes; these induced, respectively, a significant increase, in {E}ubacteria and {R}oseobacter and no significant changes in {G}ammaproteobacteria concentrations from the investigated natural environment. {W}e confirmed the eukaryotic probes original conditions, and propose the {E}uk1209+{NC}hlo01+{C}hlo02 probe mix to target the largest picoeukaryotic diversity. {E}xperiences acquired through these investigations leads us to propose the use of seven steps protocol for complete {FISH} probe specificity check-up to improve data quality in environmental studies.}, keywords = {{TSA}-{FISH} ; probes ; specificity ; marine ; prokaryotes ; eukaryotes ; oligonucleotide ; {CARD}-{FISH}}, booktitle = {}, journal = {{F}rontiers in {M}icrobiology}, volume = {8}, numero = {}, pages = {art. 854 [13 p.]}, ISSN = {1664-302{X}}, year = {2017}, DOI = {10.3389/fmicb.2017.00854}, URL = {https://www.documentation.ird.fr/hor/fdi:010070083}, }