A new semi-nested PCR protocol to amplify large 18S rRNA gene fragments for PCR-DGGE analysis of soil fungal communities

Oros-Sichler, Miruna; Gomes, Newton C. M.; Neuber, Gabriele; Smalla, Kornelia

doi:10.1016/j.mimet.2005.06.014

Cited by 76 publications

(52 citation statements)

References 27 publications

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“…Application of PCR in amplifying 18S rRNA genes has contributed to our better understanding of evolutionary relationship between fungi (White et al, 1990;Wilmotte et al, 1993) and also enhanced our ability to describe fungal communities with respect to environmental factors and perturbations (Kowalchuk et al, 1997;Malosso et al, 2006;Oros-Sichler et al, 2006;Jumpponen, 2007). DGGE analysis based on 18S rRNA fragments has been used to reveal fungal communities in many terrestrial natural habitats (Kowalchuk et al, 1997;Borneman and Hartin, 2000;Vainio and Hantula, 2000;May et al, 2001;Brodie et al, 2003;Nikolcheva et al, 2003), but its application for investigating fungal communities in marine environments remains rare (Pang and Mitchell, 2005;Gao et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

Molecular characterization of the spatial diversity and novel lineages of mycoplankton in Hawaiian coastal waters

2009

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Microbial community diversity and composition have critical biogeochemical roles in the functioning of marine ecosystems. Large populations of planktonic fungi exist in coastal ocean waters, yet their diversity and role in carbon and nutrient cycling remain largely unknown. Lack of information on critical functional microbial groups limits our understanding of their ecological roles in coastal oceans and hence our understanding of its functioning in the ocean's carbon and nutrient cycles. To address this gap, this study applied the molecular approach denaturing gradient gel electrophoresis (DGGE) coupled with clone library construction to investigate mycoplankton communities in Hawaiian coastal waters. Mycoplankton communities displayed distinct lateral and vertical variations in diversity and composition. Compared with the open ocean, surface (o100 m) near-shore waters had the greatest diversity and species richness of mycoplankton, whereas no differences were found among stations at depths below 150 m. Vertical diversity profiles in the coastal waters suggested that diversity and species richness were positively correlated to phytoplankton biomass in the coastal waters, but not in offshore waters. A total of 46 species were identified and belonging to two phyla Basidiomycota and Ascomycota, with the basidiomycetes as the dominant group (n ¼ 42). The majority (n ¼ 27) of the basidiomycetes are novel phylotypes showing less than 98% identity in the 18S rRNA gene with any sequence in GenBank. This study provides insight into mycoplankton ecology and is the first molecular analysis of planktonic fungi in the oceans. The ISME Journal (

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Section: Discussionmentioning

confidence: 99%

Molecular characterization of the spatial diversity and novel lineages of mycoplankton in Hawaiian coastal waters

2009

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“…In microbial ecology, culture dependent methods have characterised soil fungal communities but these detect only a fraction (ca 1%) of the total populations [22,23].…”

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confidence: 99%

Soil fungal community shift evaluation as a potential cadaver decomposition indicator

Chimutsa

Olakanye

Thompson

et al. 2015

Forensic Science International

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“…Primer set U comprised primers U1 and U2, and also targeted the 26-28S rRNA gene, yielding amplicons of~260 bp (Sandhu et al, 1995). Primer set E comprised primer NS1 (White et al, 1990), EF3 (Smit et al, 1999) and FR1 (Vainio & Hantula, 2000), which were used in a semi-nested PCR to amplify the V1-V9 region of the 18S rRNA gene, yielding a product of~1650 bp (Oros-Sichler et al, 2006). PCR was performed in 0.2 ml tubes using a Hybaid Px2 thermal cycler.…”

Section: Methodsmentioning

confidence: 99%

“…Semi-nested PCR was performed by modification of a published procedure (Oros-Sichler et al, 2006). The first amplification step involved 25 PCR cycles with the NS1 and EF3 primers.…”

Section: Methodsmentioning

confidence: 99%

“…DGGE based on 16S and 26-28S rRNA gene sequences has been used to characterize complex microbial communities, including those in saliva, dental plaque and plaque microcosms (Ledder et al, 2006;Li et al, 2006;Rasiah et al, 2005;Zijnge et al, 2003). It has also been applied to monitor fungal communities in complex ecosystems such as soil, and in wine and food fermentations (Cocolin et al, 2000;Meroth et al, 2003;Muccilli et al, 2011;Oros-Sichler et al, 2006;Vilela et al, 2010). The use of this technique to identify yeasts in the oral cavity has potential both as a diagnostic tool and as a method to advance our understanding of these complex ecosystems.…”

Section: Introductionmentioning

confidence: 99%

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Use of denaturing gradient gel electrophoresis for the identification of mixed oral yeasts in human saliva

Weerasekera

Sissons

Wong

et al. 2013

Journal of Medical Microbiology

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A PCR-denaturing gradient gel electrophoresis (DGGE) method was established for the simultaneous presumptive identification of multiple yeast species commonly present in the oral cavity. Published primer sets targeting different regions of the Saccharomyces cerevisiae 26-28S rRNA gene (denoted primer sets N and U) and the 18S rRNA gene (primer set E) were evaluated with ten Candida and four non-Candida yeast species, and twenty Candida albicans isolates. Optimized PCR-DGGE conditions using primer set N were applied to presumptively identify, by band matching, yeasts in the saliva of 25 individuals. Identities were confirmed by DNA sequencing and compared with those using CHROMagar Candida culture. All primer sets yielded detectable DGGE bands for all species tested. Primer set N yielded mainly single bands and could distinguish all species examined, including differentiating Candida dubliniensis from C. albicans. Primer set U was less discriminatory among species but yielded multiple bands that distinguished subspecies groups within C. albicans. Primer set E gave poor yeast discrimination. DGGE analysis identified yeasts in 17 of the 25 saliva samples. Six saliva samples contained two yeast species: three contained C. albicans and three C. dubliniensis. C. dubliniensis was present alone in one saliva sample (total prevalence 16 %). CHROMagar culture detected yeasts in 16 of the yeast-containing saliva samples and did not enable identification of 7 yeast species identified by DGGE. In conclusion, DGGE identification of oral yeast species with primer set N is a relatively fast and reliable method for the simultaneous presumptive identification of mixed yeasts in oral saliva samples.

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A new semi-nested PCR protocol to amplify large 18S rRNA gene fragments for PCR-DGGE analysis of soil fungal communities

Cited by 76 publications

References 27 publications

Molecular characterization of the spatial diversity and novel lineages of mycoplankton in Hawaiian coastal waters

Molecular characterization of the spatial diversity and novel lineages of mycoplankton in Hawaiian coastal waters

Soil fungal community shift evaluation as a potential cadaver decomposition indicator

Use of denaturing gradient gel electrophoresis for the identification of mixed oral yeasts in human saliva

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