Development of molecular tools for the detection of freshwater diatoms.
This study was undertaken to develop a reliable and reproducible procedure for the detection and quantitative determination of diatoms in environmental samples. A comparative study of seven different DNA extraction kits was carried out to establish conditions for analysis of diatom containing samples. The best performers were identified using both standard and real-time PCR. We show that the yield of diatom DNA is generally quite low when using commercially available extraction kits; in addition, a new protocol was devised to obtain samples suitable for DNA amplification without the need to perform all the steps required for DNA extraction. This method was tested on environmental samples spiked, in a wide range of total cell mass, with the rarely occurring diatom Neidium affine together with a highly species-specific oligonucleotide designed on the small subunit (SSU) rRNA gene. Thus, we propose a fast and effective procedure that, combined with the use of species-specific oligonucleotide probes can detect minute amounts of a spiked diatom within a complex diatom community. This study provides experimental conditions for a fast and accurate detection of diatoms, and demonstrates the feasibility of the use of molecular tools in the evaluation of water quality.
Department of Biosciences and Biotechnology, University of Camerino, Via Gentile III da Varano, 62032 Camerino, Italy.
Thi Nhu Mai
TN
Nguyen
Marco
M
Berzano
Claudio O
CO
Gualerzi
Roberto
R
Spurio
D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D017377:Diatoms / Q000235:genetics* / Q000302:isolation & purification*; D005618:Fresh Water / Q000382:microbiology*; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D015345:Oligonucleotide Probes; D004247:DNA