Comparison of two DNA extraction methods widely used in aquatic microbial ecology

Mateus-Barros, Erick; Meneghine, Aylan Kener; Bagatini, Inessa Lacativa; Fernandes, Camila Cesário; Kishi, Luciano Takeshi; Vieira, Armando Augusto Henriques; Sarmento, Hugo

doi:10.1016/j.mimet.2019.02.005

Cited by 8 publications

(6 citation statements)

References 25 publications

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“…The same analysis using a higher clustering resolution (ASVs) yielded slightly different results: the bimodality remained significant, while the bimodal hump was weaker and only three ASVs were core. The inaccuracy of molecular methods used to access the microbial biodiversity, from DNA extraction [29], amplification [47], and sequencing errors [48], could bias the interpretation of ecological processes at a high clustering resolution level, such as ASVs. Actually, both methods used to determine taxonomic units are prone to criticism, as they define taxonomy based on an arbitrary threshold value (97% similarity for OTUs; 100% similarity for ASVs), OTU does not consider different diversification rates for each bacterial phylogenetic group, and ASV may artificially overestimate diversity [48].…”

Section: Discussionmentioning

confidence: 99%

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Local and Geographic Factors Shape the Occupancy-Frequency Distribution of Freshwater Bacteria

et al. 2020

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Species prevalence across the landscape is related to their local abundance, which is a result of deterministic and stochastic processes that select organisms capable of recolonizing sites where they were once extinct, a process known as the rescue effect. The occupancy-frequency distribution (OFD) describes these patterns and has been extensively used to understand organism's distribution but has been poorly tested on microorganisms. In order to test OFD on freshwater bacteria, we collected data from 60 shallow lakes distributed across a wide area in southeastern Brazil, to determine the bacterial operational taxonomic units (OTUs) that were present in all sites (core) and at only one site (satellite). Then, we analyzed the spatial abundance distributions of individual OTUs to understand the influence of local abundances on regional occupancy patterns. Finally, we tested the environmental factors that influenced occupancy and abundance. We found a significant bimodal OFD for freshwater bacteria using both OTUs (97% clustering) and amplicon sequence variants (ASVs, unique sequences), with 13 core OTUs and 1169 satellite OTUs, but only three core ASVs. Core organisms had a bimodal or gamma abundance distribution. The main driver of the core community was pH, while nutrients were key when the core community was excluded and the rest of the community (mild and satellite taxa) was considered. This study demonstrates the close relationship between local environmental conditions and the abundance and dispersion of microorganisms, which shapes their distribution across the landscape.

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Section: Discussionmentioning

confidence: 99%

“…A detailed description of the molecular procedure and bioinformatics were provided by Mateus-Barros and collaborators [29]. Briefly, we used one half of a filter to extract the bacterial DNA from each sample with phenol-chloroform.…”

Section: Sampling Procedures Molecular Analysis and Bioinformatics For Microbial Communitiesmentioning

confidence: 99%

Local and Geographic Factors Shape the Occupancy-Frequency Distribution of Freshwater Bacteria

et al. 2020

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“…Method 4 was an in-house extraction method, based on the standard phenol-chloroform-isoamyl alcohol protocol [18] with certain modifications. Briefly, 1 mL of each water and the suspension of food was boiled in a water bath for 10 min and then put into ultrasonic bath for additional 10 min in order to cause cell membrane lysis without the use of a chaotropic agent (e.g., phenol).…”

Section: Methodsmentioning

confidence: 99%

A comparison of different DNA extraction methods and molecular techniques for the detection and identification of foodborne pathogens

2021

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<abstract> <p>Foodborne infections continue to plague Europe. Food safety monitoring is in crisis as the existing techniques for detecting pathogens do not keep up with the global rising of food production and consumption. Thus, the development of innovative techniques for detecting and identifying pathogenic bacteria has become critical. The aim of the present study was firstly to develop an innovative simple and low cost method of extracting bacterial DNA from contaminated food and water samples with <italic>Salmonella enteric</italic> subsp. <italic>enteric</italic> serovar Typhimurium and <italic>Listeria monocytogenes</italic> and its comparison with two commercial DNA extraction kits (Qiagen, Macherey-Nagel). Finally, pathogens' detection using two molecular techniques (PCR-electrophoresis, LAMP), in order to evaluate the best combination of DNA extraction and identification based on their sensitivity, cost, rapidity and simplicity. Considering the above criteria, among them, best was proved an in-house bacterial DNA extraction method, based on the chloroform-isoamyl alcohol protocol, with certain modifications. This technique showed statistically similar results in terms of sensitivity, compared to the commercial kits, while at the same time maintained high rapidity and much lower cost. Lastly, between the molecular techniques, LAMP was found more promising considering its simplicity, high rapidity and sensitivity. Conclusively, the in-house DNA extraction method along with the LAMP technique, was proven to be the best among the presented combinations.</p> </abstract>

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“…The DNA samples were extracted using the MoBio PowerSoil DNA Isolation Kit (Mo-Bio, Solana Beach, CA, USA) [24]. The amplification conditions for qPCR and primers for the genes are listed in the Supplementary Materials.…”

Section: Qpcr Assay and High-throughput Sequencingmentioning

confidence: 99%

The Substitution of Sphagnum for Peat as a Culture Substrate Reduces N2O Emissions from Vegetable Production Systems

Liang,

Wang,

Zhang

et al. 2024

Agronomy

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Peat-based substrates have been widely used in greenhouse vegetable production (GVP). However, peat is a non-renewable resource, and there is a problem with N2O emissions when it is used in greenhouse vegetable production due to the application of large quantities of nutrient solutions. Sphagnum (SP) is a precursor substance and a renewable resource for peat formation, and it has good physical and chemical properties. However, there has been no study on the effect of using sphagnum to replace peat in greenhouse vegetable production on N2O emissions. Therefore, this study used a peat substrate as the control treatment (CK), with sphagnum replacing peat at 25% (25SP), 50% (50SP), 75% (75SP), and 100% (100SP) in six treatment groups. Moreover, lettuce was used as the experimental subject in potting experiments, and the physicochemical properties, N2O emissions, N2O isotope δ value, and N2O-related microbial activity and community structures were determined using different treatments. Compared with the CK treatment, the 25SP treatment significantly reduced N2O emissions by 55.35%, while the 75SP treatment significantly increased N2O emissions by 67.76%. The 25SP treatment reduced N2O to N2 to the highest extent and demonstrated the lowest contribution of fungal denitrification (FD) and bacterial nitrification (BN) processes, thereby resulting in lower N2O emissions. In contrast, NH4+ and NO3− were the main substrates for N2O emissions; the 75SP treatment had higher NH4+ and NO3− contents and a lower relative abundance of the nosZ gene, thereby resulting in higher N2O emissions. In addition, N2O production and reduction were dominated by bacterial denitrification for all treatments. Thus, this study analyzed the community composition of denitrifying bacterial genera and their association with physicochemical properties. The results indicated that the dominant denitrifying genus in the peat substrate was Rhodanobacter and that sphagnum replacement reduced the relative abundance of Rhodanobacter. The dominant genus was Massilia at 100% sphagnum replacement. More importantly, Rhodanobacter was correlated with C/N and electrical conductivity (EC), whereas Massilia was affected by NH4+ and the water-filled pore space (WFPS). Therefore, different denitrification-dominant genera were affected by different environmental factors, which indirectly affected N2O emission. In summary, the 25SP treatment was able to improve nitrogen use efficiency and had no significant effect on lettuce yield. Therefore, 25% sphagnum replacement is the most suitable percentage for peat replacement.

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Comparison of two DNA extraction methods widely used in aquatic microbial ecology

Cited by 8 publications

References 25 publications

Local and Geographic Factors Shape the Occupancy-Frequency Distribution of Freshwater Bacteria

Local and Geographic Factors Shape the Occupancy-Frequency Distribution of Freshwater Bacteria

A comparison of different DNA extraction methods and molecular techniques for the detection and identification of foodborne pathogens

The Substitution of Sphagnum for Peat as a Culture Substrate Reduces N2O Emissions from Vegetable Production Systems

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