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IDENTIFICATION OF THE CLASS PRYMNESIOPHYCEAE AND THE GENUS PHAEOCYSTIS WITH RIBOSOMAL RNA–TARGETED NUCLEIC ACID PROBES DETECTED BY FLOW CYTOMETRY 1
Author(s) -
Lange Martin,
Guillou Laure,
Vaulot Daniel,
Simon Nathalie,
Amann Rudolf I.,
Ludwig Wolfgang,
Medlin Linda K.
Publication year - 1996
Publication title -
journal of phycology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.85
H-Index - 127
eISSN - 1529-8817
pISSN - 0022-3646
DOI - 10.1111/j.0022-3646.1996.00858.x
Subject(s) - biology , autofluorescence , ribosomal rna , flow cytometry , nucleic acid , molecular probe , fluorescence , fluorescein , fluorescein isothiocyanate , fluorescence in situ hybridization , microbiology and biotechnology , hybridization probe , biochemistry , dna , gene , physics , quantum mechanics , chromosome
Target regions specific for the class Prymnesiophyceae and the genus Phaeocystis (Har.) Lag. were identified from 18S ribosomal RNA coding regions, and two complementary probes were designed (PRYMN01 and PHAEO01). Detection of whole cells hybridized with these probes labeled with fluorescein isothiocyanate was difficult using epifluorescence microscopy because autofluorescence of the chlorophylls seriously interfered with the fluorescence of the probes. In contrast, flow cytometry proved very useful to detect and quantify the fluorescence of the hybridized cells. Hybridization conditions were optimized, especially with respect to formamide concentration. Both probes were tested on a large array of both target and nontarget strains. Positive and negative controls were also analyzed. Specificity was tested by adding a competing nonlabeled probe. Whereas probe PHAEO01 seems to have good specificity, probe PRYMN01 appeared less specific and must be used with stringent positive and negative controls.