Improved purification and PCR amplification of DNA from environmental samples

Arbeli, Ziv; Fuentes, Cilia L.

doi:10.1111/j.1574-6968.2007.00764.x

Cited by 93 publications

(72 citation statements)

References 31 publications

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“…LAMP amplification of bla NDM-1 failed under the standard conditions. It is reported that, amplification of DNA improves in the presence of additives like Dimethly sulfoxide (DMSO), PEG [21][22][23]. Therefore, the effect of DMSO, PEG 4KDa and PEG 8KDa on the efficiency of LAMP was assessed.…”

Section: Resultsmentioning

confidence: 99%

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Molecular Detection of New Delhi Metallo-Beta-Lactamase-1 (NDM-1) Positive Bacteria from Environmental and Drinking Water Samples by Loop Mediated Isothermal Amplification of bla NDM-1

et al. 2015

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New Delhi metallo-b-lactamase-1 gene (bla NDM-1 ) codes for New Delhi metallo-beta-lactamase-1 (NDM-1) enzyme that cleaves the amide bond of b-lactam ring, and provides resistance against major classes of blactam antibiotics. Dissemination of the plasmid borne bla NDM-1 through horizontal gene transfer is a potential threat to the society. In this study, a rapid non-culture method for detecting NDM-1 positive bacteria was developed by Loop Mediated Isothermal Amplification (LAMP) of bla NDM-1 . Sensitivity of this method was found to be one femtogram of plasmid DNA, which translates into 2.6-25.8 copies depending on the size of the plasmid DNA. This method was applied to detect NDM-1 positive bacteria in 81 water samples that were collected from environmental and drinking water sources. NDM-1 positive bacteria were detected in three drinking water samples by LAMP but not by PCR. These three samples were collected from the water sources that were treated with chlorine for decontamination before public distribution. NDM-1 positive bacteria were not detected in lake water samples or in the samples that were collected from the water sources that were purified by reverse osmosis before public distribution. Detection of NDM-1 positive bacteria using LAMP was found to be safe, sensitive and rapid for screening large number of samples from diverse sources. This method could be developed as on-field detection kit by using fluorescent dyes to visualize the amplified bla NDM-1 gene.

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Section: Resultsmentioning

confidence: 99%

“…While we have used plasmid DNA as template Liu et al [20] have used genomic DNA, which is relatively more complex. Further, we have used 5 % DMSO, which is reported to enhance primer annealing and amplification efficiency with GC rich templates [21][22][23].…”

Section: Comparison Of Lamp and Pcrmentioning

confidence: 99%

Molecular Detection of New Delhi Metallo-Beta-Lactamase-1 (NDM-1) Positive Bacteria from Environmental and Drinking Water Samples by Loop Mediated Isothermal Amplification of bla NDM-1

et al. 2015

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“…Several studies have addressed this issue, providing strategies for the removal of humic and fulvic acids during the extraction of nucleic acids (1). Suggested methodology includes Sephadex spin columns and polyethylene glycol (PEG) precipitation of nucleic acids (11). Continued inhibition after extract purification might be related to the high concentrations of enzyme-inhibiting phenolic compounds, particularly in anoxic soils such as peat (12,13).…”

mentioning

confidence: 99%

Metatranscriptomic Analysis of Arctic Peat Soil Microbiota

Tveit

Urich

Svenning

2014

Appl Environ Microbiol

146

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c Recent advances in meta-omics and particularly metatranscriptomic approaches have enabled detailed studies of the structure and function of microbial communities in many ecosystems. Molecular analyses of peat soils, ecosystems important to the global carbon balance, are still challenging due to the presence of coextracted substances that inhibit enzymes used in downstream applications. We sampled layers at different depths from two high-Arctic peat soils in Svalbard for metatranscriptome preparation. Here we show that enzyme inhibition in the preparation of metatranscriptomic libraries can be circumvented by linear amplification of diluted template RNA. A comparative analysis of mRNA-enriched and nonenriched metatranscriptomes showed that mRNA enrichment resulted in a 2-fold increase in the relative abundance of mRNA but biased the relative distribution of mRNA. The relative abundance of transcripts for cellulose degradation decreased with depth, while the transcripts for hemicellulose debranching increased, indicating that the polysaccharide composition of the peat was different in the deeper and older layers. Taxonomic annotation revealed that Actinobacteria and Bacteroidetes were the dominating polysaccharide decomposers. The relative abundances of 16S rRNA and mRNA transcripts of methanogenic Archaea increased substantially with depth. Acetoclastic methanogenesis was the dominating pathway, followed by methanogenesis from formate. The relative abundances of 16S rRNA and mRNA assigned to the methanotrophic Methylococcaceae, primarily Methylobacter, increased with depth. In conclusion, linear amplification of total RNA and deep sequencing constituted the preferred method for metatranscriptomic preparation to enable high-resolution functional and taxonomic analyses of the active microbiota in Arctic peat soil.

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“…These observations suggest that our DNA-extraction method does not always completely clean the DNA from potential PCR inhibitors present in the sediment trap material such as polysaccharides. However, it is not uncommon that PCR-amplification from sediment samples is hampered by inhibitors present in environmental samples e.g., from river sediments (Arbeli and Fuentes, 2007). Thus, individual optimizations of amplification protocols for single samples might be necessary for future PCR-based molecular analyses of long-term sediment trap samples.…”

Section: Mercury Chloride Preservationmentioning

confidence: 99%

Protist Communities in Moored Long-Term Sediment Traps (Fram Strait, Arctic)–Preservation with Mercury Chloride Allows for PCR-Based Molecular Genetic Analyses

et al. 2017

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Here we present a pilot study demonstrating, that preservation with mercury chloride allows the application of PCR-based molecular methods for the characterization of marine protist communities collected with moored long-term sediment traps. They can provide information on pelagic protist communities by collecting sinking plankton from the upper water column all year-round, even in remote polar oceans. Assessment of small protist species from the nano-and picoplankton fractions in sedimented material by microscopy is extremely challenging or almost impossible. Hence, comprehensive studies of variability in protist community composition in moored long-term sediment traps are scarce. Considering that marine nano-and picoeukaryotes are ecologically very important, new approaches are urgently needed to investigate protists in the smallest size-fractions of moored long-term sediment trap samples. We applied the quick and cost-effective the door for molecular genetic analyses of long-term sediment trap samples, and that PCR-based molecular methods have a strong potential to become an important tool for comprehensive taxonomic analyses of protist-and bacterial communities in moored long-term sediment traps.

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Improved purification and PCR amplification of DNA from environmental samples

Cited by 93 publications

References 31 publications

Molecular Detection of New Delhi Metallo-Beta-Lactamase-1 (NDM-1) Positive Bacteria from Environmental and Drinking Water Samples by Loop Mediated Isothermal Amplification of bla NDM-1

Molecular Detection of New Delhi Metallo-Beta-Lactamase-1 (NDM-1) Positive Bacteria from Environmental and Drinking Water Samples by Loop Mediated Isothermal Amplification of bla NDM-1

Metatranscriptomic Analysis of Arctic Peat Soil Microbiota

Protist Communities in Moored Long-Term Sediment Traps (Fram Strait, Arctic)–Preservation with Mercury Chloride Allows for PCR-Based Molecular Genetic Analyses

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