Contamination of diverse<i>nifH</i>and<i>nifH</i>-like DNA into commercial PCR primers

Goto, Masahiro; Ando, Sugihiro; Hachisuka, Yusuke; Yoneyama, T.

doi:10.1016/j.femsle.2005.03.042

Cited by 40 publications

(33 citation statements)

References 33 publications

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“…Diazotrophs are typically found in low abundance in marine systems, and a nested PCR protocol (455 amplification cycles) is required to amplify the nifH gene. Clone libraries generated using these amplicons may contain heterotrophic nifH sequences amplified from organisms or nucleic acids present in PCR reagents (Kulakov et al, 2002;Zehr et al, 2003a;Goto et al, 2005;Farnelid et al, 2009), high-purity water systems (Matsuda et al, 1996) or sampling equipment. It is insufficient to run negative controls, as amplification of contaminant nifH is sporadic and difficult to reproduce.…”

Section: Resultsmentioning

confidence: 99%

“…Although most of these are Cluster I sequences, a Cluster III sequence has been reported (AB198377; Goto et al, 2005). CV9 has 99% nucleotide similarity to a known PCR contaminant, AB198391 (Goto et al, 2005), and is presumed to be a contaminant. Other sequences may be contaminants based on Figure 1 Neighbor-joining tree of partial nifH cyanobacterial sequences obtained from Cape Verde transcripts.…”

Section: Resultsmentioning

confidence: 99%

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Nitrogen fixation and nitrogenase (nifH) expression in tropical waters of the eastern North Atlantic

et al. 2011

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Expression of nifH in 28 surface water samples collected during fall 2007 from six stations in the vicinity of the Cape Verde Islands (north-east Atlantic) was examined using reverse transcriptionpolymerase chain reaction (RT-PCR)-based clone libraries and quantitative RT-PCR (RT-qPCR) analysis of seven diazotrophic phylotypes. Biological nitrogen fixation (BNF) rates and nutrient concentrations were determined for these stations, which were selected based on a range in surface chlorophyll concentrations to target a gradient of primary productivity. BNF rates greater than 6 nmolN l À1 h À1 were measured at two of the near-shore stations where high concentrations of Fe and PO 4 3À were also measured. Six hundred and five nifH transcripts were amplified by RT-PCR, of which 76% are described by six operational taxonomic units, including Trichodesmium and the uncultivated UCYN-A, and four non-cyanobacterial diazotrophs that clustered with uncultivated Proteobacteria. Although all five cyanobacterial phylotypes quantified in RT-qPCR assays were detected at different stations in this study, UCYN-A contributed most significantly to the pool of nifH transcripts in both coastal and oligotrophic waters. A comparison of results from RT-PCR clone libraries and RT-qPCR indicated that a c-proteobacterial phylotype was preferentially amplified in clone libraries, which underscores the need to use caution interpreting clone-library-based nifH studies, especially when considering the importance of uncultivated proteobacterial diazotrophs.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Nitrogen fixation and nitrogenase (nifH) expression in tropical waters of the eastern North Atlantic

et al. 2011

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“…One drawback of the nested PCR approach is the high susceptibility to contamination through handling or presence of nifH genes in PCR reagents (Zehr et al, 2003a;Goto et al, 2005). To circumvent this potential risk, nifH phylotypes may be quantified using QPCR (Hewson et al, 2007).…”

Section: Distribution and Abundances Of Nifh Phylotypes And Transcriptsmentioning

confidence: 99%

Active nitrogen-fixing heterotrophic bacteria at and below the chemocline of the central Baltic Sea

et al. 2013

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The Baltic Sea receives large nitrogen inputs by diazotrophic (N 2 -fixing) heterocystous cyanobacteria but the significance of heterotrophic N 2 fixation has not been studied. Here, the diversity, abundance and transcription of the nifH fragment of the nitrogenase enzyme in two basins of the Baltic Sea proper was examined. N 2 fixation was measured at the surface (5 m) and in anoxic water (200 m). Vertical sampling profiles of 410 and o10 lm size fractions were collected in 2007, 2008 and 2011 at the Gotland Deep and in 2011 in the Bornholm Basin. Both of these stations are characterized by permanently anoxic bottom water. The 454-pyrosequencing nifH analysis revealed a diverse assemblage of nifH genes related to alpha-, beta-and gammaproteobacteria (nifH cluster I) and anaerobic bacteria (nifH cluster III) at and below the chemocline. Abundances of genes and transcripts of seven diazotrophic phylotypes were investigated using quantitative polymerase chain reaction revealing abundances of heterotrophic nifH phylotypes of up to 2.1 Â 10 7 nifH copies l À 1 . Abundant nifH transcripts (up to 3.2 Â 10 4 transcripts l À 1 ) within nifH cluster III and co-occurring N 2 fixation (0.44 ± 0.26 nmol l À 1 day À 1 ) in deep water suggests that heterotrophic diazotrophs are fixing N 2 in anoxic ammonium-rich waters. Our results reveal that N 2 fixation in the Baltic Sea is not limited to illuminated N-deplete surface waters and suggest that N 2 fixation could also be of importance in other suboxic regions of the world's oceans. The ISME Journal (2013Journal ( ) 7, 1413Journal ( -1423 doi:10.1038/ismej.2013 published online 28 February 2013 Subject Category: microbial ecology and functional diversity of natural habitats

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“…The efficiency of the real time PCR reactions were 86.8%±0.25, and the specificity of the reactions was confirmed by gel electrophoresis where all samples produced the predicted 361 bp PCR fragment (data not shown). Finally, water was used instead of templates as a negative control to exclude that primers and PCR buffers used in this study were contaminated with nifH genes, as indicated by a study on commercial PCR primers and polymerases (Goto et al 2005). Samples were analysed in three separate runs, with two replicates of each sample in each run.…”

Section: Primer Selectionmentioning

confidence: 99%

Diazotrophic diversity, nifH gene expression and nitrogenase activity in a rice paddy field in Fujian, China

et al. 2009

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The diazotrophic communities in a rice paddy field were characterized by a molecular polyphasic approach including DNA/RNA-DGGE fingerprinting, real time RT-PCR analysis of nifH gene and the measurement of nitrogen fixation activities. The investigation was performed on a diurnal cycle and comparisons were made between bulk and rhizosphere / root soil as well as between fertilized / unfertilized soils. Real time RT-PCR showed no significant difference in the total quantity of nifH expression under the conditions investigated. The functional diversity and dynamics of the nifH gene expressing diazotroph community investigated using RT-PCR-DGGE revealed high diurnal variations, as well as variation between different soil types. Most of the sequence types recovered from the DGGE gels and clone libraries clustered within nifH Cluster I and III (65 different nifH sequences in total). Sequence types most similar to Azoarcus spp., Metylococcus spp., Rhizobium spp., Methylocystis spp., Desulfovibrio spp., Geobacter spp., Chlorobium spp., were abundant and indicate that these species may be responsible for the observed diurnal variation in the diazotrophic community structure in these rice field samples. Previously described diazotrophic cyanobacterial genera in rice fields, such as Nostoc and Cyanothece, were present in the samples but not detectable in RT-PCR assays.

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Contamination of diversenifHandnifH-like DNA into commercial PCR primers

Cited by 40 publications

References 33 publications

Nitrogen fixation and nitrogenase (nifH) expression in tropical waters of the eastern North Atlantic

Nitrogen fixation and nitrogenase (nifH) expression in tropical waters of the eastern North Atlantic

Active nitrogen-fixing heterotrophic bacteria at and below the chemocline of the central Baltic Sea

Diazotrophic diversity, nifH gene expression and nitrogenase activity in a rice paddy field in Fujian, China

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