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Materials 2018, 11(7), 1129;

Specificities and Efficiencies of Primers Targeting Candidatus Phylum Saccharibacteria in Activated Sludge

Department of Civil and Environmental Engineering, Graduate School of Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashihiroshima 739-8527, Japan
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima 739-8530, Japan
Author to whom correspondence should be addressed.
Received: 7 May 2018 / Revised: 28 June 2018 / Accepted: 2 July 2018 / Published: 3 July 2018
(This article belongs to the Special Issue Fluorescent Sensors for Selective Detection)
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Candidatus Saccharibacteria is a well-described candidate phylum that has not been successfully isolated. Nevertheless, its presence was suggested by 16S rRNA gene sequencing data, and it is frequently detected in natural environments and activated sludge. Because pure culture representatives of Candidatus Saccharibacteria are lacking, the specificity of primers for the determination of their abundance and diversity should be carefully evaluated. In this study, eight Candidatus Saccharibacteria-specific primers were selected from previous studies and evaluated for their coverage against a public database, annealing temperature of the combined primer sets, as well as their utilization to determine the detection frequencies and phylogenetic diversity by cloning analysis, and in quantification by quantitative polymerase chain reaction (PCR). Among the eight primers, four primers (TM7314F, TM7580F, TM7-910R, and TM7-1177R) showed high coverage. Cloning analysis showed that four primer sets (TM7314F and TM7-910R, TM7314F and TM7-1177R, TM7580F and TM7-910R, and TM7580F and TM7-1177R) yielded high detection frequencies for Candidatus Saccharibacteria in activated sludge from a wastewater treatment plant in Higashihiroshima City, Japan. Quantitative PCR results indicated that the primer set containing TM7314F and TM7-910R was superior for the specific detection of Candidatus Saccharibacteria in activated sludge. View Full-Text
Keywords: Candidatus Saccharibacteria; primer evaluation; primer specificity; quantitative PCR; probe Candidatus Saccharibacteria; primer evaluation; primer specificity; quantitative PCR; probe

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Takenaka, R.; Aoi, Y.; Ozaki, N.; Ohashi, A.; Kindaichi, T. Specificities and Efficiencies of Primers Targeting Candidatus Phylum Saccharibacteria in Activated Sludge. Materials 2018, 11, 1129.

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