Preparation of DNA Suitable for PCR Amplification from Fresh or Fixed Single Dinoflagellate Cells

Marı́n, Irma; Aguilera, Ángeles; Reguera, B.; Abad, José Pascual

doi:10.2144/01301st05

Cited by 52 publications

(44 citation statements)

References 9 publications

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“…Successful single-cell PCR from preserved plankton samples has been demonstrated for cultured strains using ethanol or methanol fixation (18), formalin or methanol fixation (13), osmium tetroxide fixation (29), and Lugol's iodine solution (14). All these methods are generally problematic for low concentrations of template DNA.…”

Section: Resultsmentioning

confidence: 99%

“…Most methods either require relatively large amounts of template DNA (i.e., cultured material, preserved tissues, or environmental DNA collected on filters or by centrifugation [18]) or amplification is limited to short fragments or both (2,4,6). It is therefore no coincidence that attempts to analyze the DNA sequence from preserved microplankton samples focused mainly on alveolate taxa, i.e., organisms presumably with a high copy number of the SSU rRNA gene (dinoflagellates [5,11,13,29]; ciliates [9]).…”

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Improved Methodology for Identification of Protists and Microalgae from Plankton Samples Preserved in Lugol's Iodine Solution: Combining Microscopic Analysis with Single-Cell PCR

Auinger

Pfandl

Boenigk

2008

Appl Environ Microbiol

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Here we introduce a method for quantitative analysis of planktonic protists and microalgae from preserved field samples combining morphological and small-subunit (SSU) rRNA gene sequence analysis. We linked a microscopic screening with PCR of single cells using field samples preserved with Lugol's iodine solution. Cells possessing a rigid cell wall were incubated with Viscozyme and subsequently with proteinase K for cell disruption; this was unnecessary for fragile cells. The addition of sodium thiosulfate to the PCR tube considerably decreased the inhibiting effect of the fixative (iodine) on the PCR and thus allowed for successful single-cell PCR even of long DNA fragments (up to as many as 3,000 base pairs). We further applied the protocol to investigate the dominant SSU rRNA genotypes in distinct flagellate morphospecies originating from different samples. We hypothesized that despite the morphological similarity, protist morphospecies in different habitats or sampled during different seasons are represented by different genotypes. Our results indicate species-specific differences: the two species Ochromonas sp. and Dinobryon divergens were represented by several different genotypes each, and for the latter species, the dominating genotype differed with habitat. In contrast, Dinobryon pediforme, Dinobryon bavaricum, and Synura sphagnicola were exclusively represented by a single genotype each, and the respective genotype was the same in different samples. In summary, our results highlight the significance of molecular variation within protist morphospecies.Linking a specific protist or microalgal small-subunit (SSU) rRNA gene sequence from environmental surveys to a specific morphotype is often problematic. Molecular surveys do not usually provide any information on the morphology of the organism (see references 19, 25, and 27 but compare with reference 10), whereas morphological surveys concentrate on preserved samples, which are usually not considered for molecular analyses (7). One main way to overcome these problems is to link sequence analysis with morphological investigations from preserved plankton samples on a per cell basis.Successful sequence analysis has already been demonstrated for preserved specimens, but it has various shortcomings. Most methods either require relatively large amounts of template DNA (i.e., cultured material, preserved tissues, or environmental DNA collected on filters or by centrifugation [18]) or amplification is limited to short fragments or both (2, 4, 6). It is therefore no coincidence that attempts to analyze the DNA sequence from preserved microplankton samples focused mainly on alveolate taxa, i.e., organisms presumably with a high copy number of the SSU rRNA gene (dinoflagellates [5,11,13,29]; ciliates [9]). Still, despite the presumably high gene copy number in the alveolates investigated so far, success with field samples is usually low.Among the most common fixatives for microalgae and protists are formaldehyde and Lugol's iodine solution (12,20,32). Formaldehyde-p...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Improved Methodology for Identification of Protists and Microalgae from Plankton Samples Preserved in Lugol's Iodine Solution: Combining Microscopic Analysis with Single-Cell PCR

Auinger

Pfandl

Boenigk

2008

Appl Environ Microbiol

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“…Unfortunately, these identification methods are lengthy and laborious, and in most cases the interpretation of the results is subjective. More recently, and based on approaches implemented for other harmful algal bloom species (34,47,58), molecular detection methods have been developed for P. piscicida and related species (6,45,53,67).…”

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confidence: 99%

Characterization of the rRNA Locus of Pfiesteria piscicida and Development of Standard and Quantitative PCR-Based Detection Assays Targeted to the Nontranscribed Spacer

Saito

Drgon

Robledo

et al. 2002

Appl Environ Microbiol

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Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 g of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.

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“…Previous studies have proved that DNA can be extracted and amplified from samples of microalgae and aquatic animals preserved with formaldehyde [16,29,38,40,45], but a relatively high rate of DNA damage was reported [10,13,47], whereas very few works have focused on DNA extraction and amplification from glutaraldehydepreserved samples. Glutaraldehyde treatments were reported to produce DNA damage in tissues of plants and fungi at rates of 0.0% to less than 0.1%, the least among cytological fixatives [13].…”

Section: Discussionmentioning

confidence: 99%

Improved Methodology for Identification of Cryptomonads: Combining Light Microscopy and PCR Amplification

Xia

Cheng²,

Zhu³

et al. 2013

J. Microbiol. Biotechnol.

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Cryptomonads are unicellular, biflagellate algae. Generally, cryptomonad cells cannot be preserved well because of their fragile nature, and an improved methodology should be developed to identify cryptomonads from natural habitats. In this study, we tried using several cytological fixatives, including glutaraldehyde, formaldehyde, and their combinations to preserve field samples collected from various waters, and the currently used fixative, Lugol's solution was tested for comparison. Results showed that among the fixatives tested, glutaraldehyde preserved the samples best, and the optimal concentration of glutaraldehyde was 2%. The cell morphology was well preserved by glutaraldehyde. Cells kept their original color, volume, and shape, and important taxonomic features such as furrow/gullet complex, ejectosomes, as well as flagella could be observed clearly, whereas these organelles frequently disappeared in Lugol's solution preserved samples. The osmotic adjustments and buffers tested could not preserve cell density significantly higher. Statistical calculation showed the cell density in the samples preserved by 2% glutaraldehyde remained stable after 43 days of the fixation procedure. In addition, DNA was extracted from glutaraldehyde preserved samples by grinding with liquid nitrogen and the 18S rDNA sequence was amplified by PCR. The sequence was virtually identical to the reference sequence, and phylogenetic analyses showed very close relationship between it and sequences from the same organism. To sum up, the present study demonstrated that 2% unbuffered glutaraldehyde, without osmotic adjustments, can preserve cryptomonads cells for identification, in terms of both light microscopy and phylogenetic analyses based on DNA sequences.

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Preparation of DNA Suitable for PCR Amplification from Fresh or Fixed Single Dinoflagellate Cells

Cited by 52 publications

References 9 publications

Improved Methodology for Identification of Protists and Microalgae from Plankton Samples Preserved in Lugol's Iodine Solution: Combining Microscopic Analysis with Single-Cell PCR

Improved Methodology for Identification of Protists and Microalgae from Plankton Samples Preserved in Lugol's Iodine Solution: Combining Microscopic Analysis with Single-Cell PCR

Characterization of the rRNA Locus of Pfiesteria piscicida and Development of Standard and Quantitative PCR-Based Detection Assays Targeted to the Nontranscribed Spacer

Improved Methodology for Identification of Cryptomonads: Combining Light Microscopy and PCR Amplification

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