A re-evaluation of dilution for eliminating PCR inhibition in soil DNA samples

Wang, Hang; Qi, Jinfeng; Xiao, Derong; Wang, Zhibao; Tian, Kang

doi:10.1016/j.soilbio.2016.12.011

Cited by 47 publications

(28 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Standard curves for fungal and bacterial nirK genes were prepared using a 10-fold dilution series of a plasmid containing target gene fragments. To remove the contamination of humid acid in soil DNA, the qPCR data were corrected following the methodology of Wang et al (2017).…”

Section: Methodsmentioning

confidence: 99%

Characterization of Fungal nirK-Containing Communities and N2O Emission From Fungal Denitrification in Arable Soils

Sheng

Xing

et al. 2019

Front. Microbiol.

View full text Add to dashboard Cite

Fungal denitrifiers play important roles in soil nitrogen cycling, but we have very limited knowledge about their distribution and functions in ecosystems. In this study, three types of arable soils were collected across different climate zones in China, including quaternary red clay soils, alluvial soils, and black soils. The composition and abundance of fungal nirK-containing denitrifiers was determined by MiSeq high-throughput sequencing and qPCR, respectively. Furthermore, a substrate-induced inhibition approach was used to explore N2O emissions from fungal denitrification. The results showed that the arable soils contained a wide range of nirK-containing fungal denitrifiers, with four orders and eight genera. Additionally, approximately 57.30% of operational taxonomic unit (OTUs) belonged to unclassified nirK-containing fungi. Hypocreales was the most predominant order, with approximately 40.51% of the total number of OTUs, followed by Sordariales, Eurotiales, and Mucorales. It was further indicated that 53% of fungal nirK OTUs were shared by the three types of soils (common), and this group of fungi comprised about 98% of the total relative abundance of the nirK-containing population, indicating that the distribution of fungal nirK-containing denitrifiers was quite homogenous among the soil types. These common OTUs were determined by multiple soil characteristics, while the composition of unique OTUs was manipulated by the specific properties of each soil type. Furthermore, fungal N2O emissions were significantly and positively correlated with fungal nirK abundance in the soils, whereas it was not clearly related to fungal nirK compositions. In conclusion, although the arable soils hosted diverse nirK-containing fungal denitrifiers, fungal nirK compositions were highly homogenous among the soil types, which could be a consequence of enduring agricultural practices. The abundance of fungal nirK-containing denitrifiers, rather than their composition, may play more significant roles in relation to N2O emission from fungal denitrification.

show abstract

Section: Methodsmentioning

confidence: 99%

Characterization of Fungal nirK-Containing Communities and N2O Emission From Fungal Denitrification in Arable Soils

Sheng

Xing

et al. 2019

Front. Microbiol.

View full text Add to dashboard Cite

show abstract

“…Genomic DNA was extracted from bulk and rhizosphere soils that had been previously stored at −80 C, using a PowerSoil DNA Isolation Kit (MoBio Laboratories). We pooled three successive extracts per sample in order to maximize DNA yields and reduce bias due to incomplete cell lysis during DNA extraction (H. Wang, Qi, Xiao, Wang, & Tian, 2017). After quality inspection, a total of 120 DNA samples (6 or 12 replicates of each soil type × 3 soil types × 2 compartments × 2 treatments) were obtained.…”

Section: Bacterial Biomassmentioning

confidence: 99%

“…DNA extraction efficiency and potential PCR reaction inhibition was taken into consideration by applying the proper dilution step for achieving accurate quantification (H. Wang, Qi, et al, 2017).…”

Section: Bacterial Biomassmentioning

confidence: 99%

Differentiating microbial taxonomic and functional responses to physical disturbance in bulk and rhizosphere soils

et al. 2020

Self Cite

View full text Add to dashboard Cite

The rhizosphere is an important hotspot of soil microbial activity, diversity, and functions. Despite being a microbial hotspot, studies have seldom addressed the differences in the response of the microbial community in bulk and rhizosphere soils to domestic animal disturbance. Here, we investigated grassland disturbance by physical uprooting behaviours of Tibetan pigs (Sus scrofa domesticus) on bacterial taxonomy and functions in three soil types: Histosols, Fluvisols, and Gleysols which are dominant on the Qinghai-Tibet Plateau. We found that after 8 years of continued disturbance, compared to the undisturbed sites, disturbance consistently reduced rhizosphere bacterial α diversity (by 34.6% on average), restructured taxonomic communities, and weakened their carbon substrate utilization capacities in all soil types. The relative abundance of the phyla Proteobacteria, Actinobacteria, and Bacteriodetes increased, but the relative abundance of Acidobacteria, Chloroflexi, and Nitrospirae decreased in the rhizosphere under disturbance. In contrast, most detected taxonomic changes in bulk soils were minor, and their changing directions were divergent among studied soil types. Compared to the undisturbed, carbon utilization potential by rhizosphere microbes decreased under disturbance and the decrease extent was stronger (by 30.4% on average) than that in bulk soil. The strengthened environmental filtering, imposed by the significant reduction of soil water-holding capacity and nutrient contents as well as the physical destruction of soil aggregates after disturbance, provided mechanistic insight into the extensive microbial restructuring within the rhizosphere. These results may have implications for recognizing changed root-microbe interactions and ecological processes in disturbed soils for better understanding soil ecology under intensified herding activities.

show abstract

“…EM6 and EM7 did not include a step of phase separation using phenol:chloroform:isoamyl alcohol and therefore had a higher chance to be ineffective at separating DNA from polysaccharides (Zhang et al, 2006), which are likely to act as PCR inhibitors (Monteiro et al, 1997;Schrader et al, 2012). A moderate level of dilution is often recommended to diminish the effect and concentration of PCR inhibitors from environmental samples (Wang et al, 2017), so we hypothesized that a second elution could improve genotyping success. Consequently, for EM6 and EM7 we tested two elutions.…”

Section: Extraction Methods 5 (Em5)mentioning

confidence: 99%

Towards high–throughput analyses of fecal samples from wildlife

Sarabia

Salado

Cornellas

et al. 2020

Anim. Biodiv. Conserv.

View full text Add to dashboard Cite

High–throughput sequencing offers new possibilities in molecular ecology and conservation studies. However, its potential has not yet become fully exploited for noninvasive studies of free–ranging animals, such as those based on feces. High–throughput sequencing allows sequencing of short DNA fragments and could allow simultaneous genotyping of a very large number of samples and markers at a low cost. The application of high throughput genotyping to fecal samples from wildlife has been hindered by several labor–intensive steps. We evaluate alternative protocols which could allow higher throughput for two of these steps: sample collection and DNA extraction. Two different field sampling and seven different DNA extraction methods are tested here on grey wolf (Canis lupus) feces. There was high variation in genotyping success rates. The field sampling method based on surface swabbing performed much worse than the extraction from a fecal fragment. In addition, there is a lot of room for improvement in the DNA extraction step. Optimization of protocols can lead to very much more efficient, cheaper and higher throughput noninvasive monitoring. Selection of appropriate markers is still of paramount importance to increase genotyping success.

show abstract

A re-evaluation of dilution for eliminating PCR inhibition in soil DNA samples

Cited by 47 publications

References 23 publications

Characterization of Fungal nirK-Containing Communities and N2O Emission From Fungal Denitrification in Arable Soils

Characterization of Fungal nirK-Containing Communities and N2O Emission From Fungal Denitrification in Arable Soils

Differentiating microbial taxonomic and functional responses to physical disturbance in bulk and rhizosphere soils

Towards high–throughput analyses of fecal samples from wildlife

Contact Info

Product

Resources

About