Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting

Watanabe, Kazuya; Kodama, Yoshio; Harayama, Shigeaki

doi:10.1016/s0167-7012(01)00220-2

Cited by 313 publications

(169 citation statements)

References 25 publications

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“…The 16S rRNA clone libraries were constructed using universal primers U27f (5 0 -AGAGTTTGATCCTGGCTCAG-3 0 ) and U1492r (5 0 -G GTTACCTTGTTACGACTT-3 0 ) (DeLong, 1992), and Systems biology for electrogenic biofilms S Ishii et al sequenced using primer U907r (5 0 -CCGYCAATT CMTTTRAGTTT-3 0 ) (Watanabe et al, 2001) as described previously (Ishii et al, 2012). The nucleotide sequences have been deposited in the GSDB/ DDBJ/EMBL/NCBI nucleotide sequence databases under accession numbers JQ654784-JQ654964, JX145950-JX146061 and KC574687-KC574877.…”

Section: Microbial Composition Analysismentioning

confidence: 99%

Microbial population and functional dynamics associated with surface potential and carbon metabolism

Ishii¹,

et al. 2013

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Microbial extracellular electron transfer (EET) to solid surfaces is an important reaction for metal reduction occurring in various anoxic environments. However, it is challenging to accurately characterize EET-active microbial communities and each member's contribution to EET reactions because of changes in composition and concentrations of electron donors and solid-phase acceptors. Here, we used bioelectrochemical systems to systematically evaluate the synergistic effects of carbon source and surface redox potential on EET-active microbial community development, metabolic networks and overall electron transfer rates. The results indicate that faster biocatalytic rates were observed under electropositive electrode surface potential conditions, and under fatty acid-fed conditions. Temporal 16S rRNA-based microbial community analyses showed that Geobacter phylotypes were highly diverse and apparently dependent on surface potentials. The well-known electrogenic microbes affiliated with the Geobacter metallireducens clade were associated with lower surface potentials and less current generation, whereas Geobacter subsurface clades 1 and 2 were associated with higher surface potentials and greater current generation. An association was also observed between specific fermentative phylotypes and Geobacter phylotypes at specific surface potentials. When sugars were present, Tolumonas and Aeromonas phylotypes were preferentially associated with lower surface potentials, whereas Lactococcus phylotypes were found to be closely associated with Geobacter subsurface clades 1 and 2 phylotypes under higher surface potential conditions. Collectively, these results suggest that surface potentials provide a strong selective pressure, at the species and strain level, for both solid surface respirators and fermentative microbes throughout the EET-active community development.

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Section: Microbial Composition Analysismentioning

confidence: 99%

Microbial population and functional dynamics associated with surface potential and carbon metabolism

Ishii¹,

et al. 2013

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“…PCR amplifications were carried out as previously described. All the primers used in this study were fully characterised in previous studies, in which they were used as primers or fluorescent in situ hybridisation (FISH) probes (Amann et al, 1990;Mobarry et al, 1996;Hovanec et al, 1998;Watanabe et al, 2001;Mohan et al, 2004;Geets et al, 2007;Smith et al, 2007). The PCR products were separated by electrophoresis on a 1% agarose gel and visualised by staining with 0.5 mg of ethidium bromide per mL of gel.…”

Section: Detection Of Nitrifying Bacteriamentioning

confidence: 99%

“…The DNA used was the same as that used for the nitrifying bacteria analyses, and PCR amplifications were carried out as previously described by (Amann et al, 1990;Trebesius et al, 2000;Watanabe et al, 2001;Fukushima et al, 2003;Qin et al, 2003;Aranda et al, 2004;Rinttilä et al, 2004;Chotár et al, 2006). The negative control was the PCR reaction containing all the components needed to perform this analysis, except DNA.…”

Section: Detection Of Potentially Pathogenic Bacteriamentioning

confidence: 99%

Characterization of the microbial community in a lotic environment to assess the effect of pollution on nitrifying and potentially pathogenic bacteria

et al. 2014

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This study aimed to investigate microbes involved in the nitrogen cycle and potentially pathogenic bacteria from urban and rural sites of the São Pedro stream. Water samples were collected from two sites. A seasonal survey of bacterial abundance was conducted. The dissolved nutrient content was analysed. PCR and FISH analysis were performed to identify and quantify microbes involved in the nitrogen cycle and potentially pathogenic bacteria. The seasonal survey revealed that the bacterial abundance was similar along the year on the rural area but varied on the urban site. Higher concentration of dissolved nutrients in the urban area indicated a eutrophic system. Considering the nitrifying microbes, the genus Nitrobacter was found, especially in the urban area, and may act as the principal bacteria in converting nitrite into nitrate at this site. The molecular markers napA, amoA, and nfrA were more accumulated at the urban site, justifying the higher content of nutrients metabolised by these enzymes. Finally, high intensity of amplicons from Enterococcus, Streptococcus, Bacteroides/Prevotella/Porphyromonas, Salmonella, S. aureus, P. aeruginosa and the diarrheagenic lineages of E. coli were observed at the urban site. These results indicate a change in the structure of the microbial community imposed by anthrophic actions. The incidence of pathogenic bacteria in aquatic environments is of particular importance to public health, emphasising the need for sewage treatment to minimise the environmental impacts associated with urbanisation.Keywords: lotic environment, urbanisation, pollution, nitrifying microbes, pathogenic bacteria.Caracterização da comunidade microbiana em um ambiente lótico para acessar o efeito da poluição em bactérias nitrificantes e potencialmente patogênicas ResumoEste estudo objetivou investigar os micro-organismos envolvidos no ciclo do nitrogênio e bactérias potencialmente patogênicas das áreas urbanas e rurais do Córrego São Pedro. Amostras de água foram coletadas dos dois locais. Um levantamento sazonal da densidade bacteriana foi realizado. O teor de nutriente dissolvido foi avaliado. As técnicas de PCR e FISH foram realizadas para identificar e quantificar os micro-organismos envolvidos no ciclo do nitrogênio e bactérias potencialmente patogênicas. O levantamento sazonal revelou que a abundância bacteriana foi semelhante ao longo do ano na área rural, porém variou na região urbana. Altas concentrações de nutrientes dissolvidos na área urbana indicaram este como um sistema eutrófico. Considerando os micro-organismos nitrificantes, o gênero Nitrobacter foi encontrado, especialmente na região urbana, e pode estar atuando como a principal bactéria convertendo nitrito em nitrato nessa área. Os marcadores moleculares napA, amoA, e nfrA foram mais acumulados na área urbana, justificando o alto teor dos nutrientes metabolizados por essas enzimas. Finalmente, alta intensidade de amplicons para Enterococcus, Streptococcus, Bacteroides/Prevotella/Porphyromonas, Salmonella, S. aureus, P. aerug...

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“…The gels were stained with 0.5 ug /ml of ethidium bromide, visualized under the UV light (Watanabe et al, 2001)and documented using a GeneSnap 4.00-Gene Genius Bio Imaging System (Syngene, Frederick, Maryland, USA).…”

Section: Analysis Of the Pcr Productsmentioning

confidence: 99%

Desulfurization of Crude Oil and Oil Products by Local Isolated Bacterial Strains

din¹

2017

Int.J.Curr.Microbiol.App.Sci

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Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting

Cited by 313 publications

References 25 publications

Microbial population and functional dynamics associated with surface potential and carbon metabolism

Microbial population and functional dynamics associated with surface potential and carbon metabolism

Characterization of the microbial community in a lotic environment to assess the effect of pollution on nitrifying and potentially pathogenic bacteria

Desulfurization of Crude Oil and Oil Products by Local Isolated Bacterial Strains

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