An optimised PCR/T-RFLP fingerprinting approach for the investigation of protistan communities in groundwater environments

• Published in: Journal of microbiological methods Vol. 75; no. 2; pp. 262 - 268
• Main Authors: Euringer, Kathrin, Lueders, Tillmann
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01-10-2008; Elsevier
• Subjects: 18S rRNA genes; Animals; Aquifer microbes; Aquifers; Biochemistry. Physiology. Immunology. Molecular biology; Biological and medical sciences; DNA Fingerprinting - methods; DNA Primers; DNA Restriction Enzymes - metabolism; Ecosystem; Eukaryota - classification; Eukaryota - genetics; Eukaryota - growth & development; Eukaryotic Cells - classification; Fresh Water - chemistry; Fresh Water - microbiology; Fundamental and applied biological sciences. Psychology; Groundwater protests; Hydrocarbons; Molecular Sequence Data; PCR selection; Polymerase Chain Reaction - methods; Polymorphism, Restriction Fragment Length; Protozoa; RNA, Ribosomal, 16S - genetics; Sequence Analysis, DNA; T-RFLP fingerprinting; Water Pollution; Water Supply; T-RFLP fingerprinting; PCR selection; Groundwater protests; Aquifer microbes; 18S rRNA genes; Ribosomal DNA; Parasite; 18S-RNA; Polymerase chain reaction; Restriction fragment length polymorphism; Gene; Ribosomal RNA; Fingerprint method; Environment
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2008.06.012

Due to the scarcity or complete absence of higher organisms, protists may represent an important higher trophic level (above Prokaryotes) in the food webs of groundwater habitats. Nevertheless, the importance of aquifer protists, especially in contaminated groundwater environments, is poorly understood. Partly, this may be due to a lack of adequate PCR and fingerprinting approaches for protists in aquifers, which can be considered low in protistan or high in non-target rRNA gene copy numbers. Therefore, we have validated the suitability of distinct eukaryote-targeted primer pairs and restriction endonucleases for T-RFLP fingerprinting of protistan communities. By
in silico predictions, and by fingerprinting, cloning and sequencing of microeukaryote amplicons from hydrocarbon-contaminated aquifer sediment DNA, we show that the Euk20f/Euk516r primer set in combination with
Bsh1236I digestion is best suited for the recovery of diverse protistan 18S rRNA lineages. In contrast to other tested primer sets, a preferred recovery of fungal and archaeal non-target amplicons was not observed. In summary, we present an optimised microeukaryote-targeted PCR/T-RFLP fingerprinting approach which may be of value for the characterisation of protistan communities in groundwater and other habitats.