Novel polymerase chain reaction primers for the specific detection of bacterial copper P‐type ATPases gene sequences in environmental isolates and metagenomic DNA

Aims: In the last decades, the worldwide increase in copper wastes release by industrial activities like mining has driven environmental metal contents to toxic levels. For this reason, the study of the biological copper‐resistance mechanisms in natural environments is important. Therefore, an appropriate molecular tool for the detection and tracking of copper‐resistance genes was developed. Methods and Results: In this work, we designed a PCR primer pair to specifically detect copper P‐type ATPases gene sequences. These PCR primers were tested in bacterial isolates and metagenomic DNA from intertidal marine environments impacted by copper pollution. As well, T‐RFLP fingerprinting of these gene sequences was used to compare the genetic composition of such genes in microbial communities, in normal and copper‐polluted coastal environments. New copper P‐type ATPases gene sequences were found, and a high degree of change in the genetic composition because of copper exposure was also determined. Conclusions: This PCR based method is useful to track bacterial copper‐resistance gene sequences in the environment. Significance and Impact of the Study: This study is the first to report the design and use of a PCR primer pair as a molecular marker to track bacterial copper‐resistance determinants, providing an excellent tool for long‐term analysis of environmental communities exposed to metal pollution.     

Application of nested polymerase chain reaction for the detection of as yet uncultured organisms of the class Actinobacteria in environmental samples

Different groups of as yet uncultured, phylogenetically deeply rooting organisms of the class Actinobacteria have been shown previously by analysis of 16S rDNA clone libraries to be present in different soil and marine environments of worldwide distribution. In order to monitor specifically the occurrence of two of the actinobacterial groups among bacterial DNA extracted from different environments, a nested polymerase chain reaction (PCR) method, aiming at the sensitive detection of 16S rDNA, was used. Analysis of a clone library generated from one of these 16S rDNA-derived, group-specific PCR products of DNA from a forest soil broadened the spectrum of putative taxa within the two phylogenetic groups. Application of this PCR system demonstrated the presence of these organisms in natural environments and in a sample of dried and moistened peat. Fluctuations in the detectability of the target groups in different samples from the same site strongly suggest that the taxon diversity of a 16S rDNA clone library must be regarded as a temporal genomic snapshot taken from this environmental community.     

Species detection using environmental DNA from water samples

The assessment of species distribution is a first critical phase of biodiversity studies and is necessary to many disciplines such as biogeography, conservation biology and ecology. However, several species are difficult to detect, especially during particular time periods or developmental stages, potentially biasing study outcomes. Here we present a novel approach, based on the limited persistence of DNA in the environment, to detect the presence of a species in fresh water. We used specific primers that amplify short mitochondrial DNA sequences to track the presence of a frog (Rana catesbeiana) in controlled environments and natural wetlands. A multi-sampling approach allowed for species detection in all environments where it was present, even at low densities. The reliability of the results was demonstrated by the identification of amplified DNA fragments, using traditional sequencing and parallel pyrosequencing techniques. As the environment can retain the molecular imprint of inhabiting species, our approach allows the reliable detection of secretive organisms in wetlands without direct observation. Combined with massive sequencing and the development of DNA barcodes that enable species identification, this approach opens new perspectives for the assessment of current biodiversity from environmental samples.     

Species-specific markers for early detection of marine invertebrate invaders through eDNA methods: Gaps and priorities in GenBank as database example

Non-indigenous species (NIS) invasions are global phenomena that are the main drivers for ecosystem change and can often drastically affect the structure and function of novel ecosystems. Early detection is of prime importance for preventing the establishment and potential dispersal of NIS, making eradication and control more efficient and less costly since the species is present at very low densities. However, species at low density generally require intensive sampling efforts to be detected.An important shortcut to reduce sampling effort is to analyze environmental DNA (eDNA). Eukaryotic organisms leave traces of their presence in the environment where they live. In environments that are difficult to access, such as aquatic environments, DNA present in the water can be extracted and amplified using PCR target markers to identify the species that inhabit therein. Using species-specific markers, the presence of target species’ DNA from water samples can be detected using PCR and simple electrophoresis in agarose gel. This is an efficient and convenient approach when the target species is known that puts the focus on detection. However, a critical challenge of this methodology is the design of appropriate species-specific markers. They must be specific to one target species, without any cross-amplification even in close species. For this purpose, a DNA database with as many different species, genetic markers and haplotypes as possible is essential for genetic marker design. The principal aim of this work is to analyze the state-of-the-art of the databases, in this case GenBank, which is the most complete database in number of markers and species, in order to assess its utility in the design of species-specific primers for invasive species early detection.     

Detection of methanogens and methanotrophs in natural environments

The role of methane as a greenhouse gas and the contribution of bacteria to the production (methanogenesis) and destruction (methane oxidation) of methane is described. Using experimental approaches based on DNA sequences identifying either methanogen-specific or methanotroph-specific gene sequences methods were developed to broaden the detection and identification of methane metabolizing bacteria in natural environments. These methods were focused on blanket bog peat but are suitable for other environments. In addition to group specific 16S rRNA DNA sequences, specific functional gene probes based on methane coenzyme reductase sequences for methanogens and methane monooxygenase sequences for methanotrophs, were developed. These sequences were used in PCR-based protocols to detect and amplify specific gene sequences from the total DNA isolated from transverse sections of blanket bog peat. This permitted the analysis of the vertical distribution of methanogen and methanotroph populations, discrimination between different sub-sets of these populations, and the identification of novel organisms not previously detected by culture-based methods.     

The utility of indels in population genetics: the Tpi intron for host race genealogy of Acrocercops transecta (Insecta: Lepidoptera)

We investigated the utility of indel data for genealogical and population genetic analyses using the Tpi intron of the leaf mining moth Acrocercops transecta (Insecta: Lepidoptera). Genealogical analyses revealed that indel data were less homoplasious than DNA sequence data and that indel data contained a sufficient signal to provide a high resolution tree that was highly congruent with the tree estimated from DNA sequences. Although some conflicts were identified in the distributions of multi-residue indels, such conflicts were especially useful for the unambiguous detection of recombinations. For the first time, we adopted a Bayesian clustering method for indel characters to infer genetic structure of the moth. We concluded that indel characters have the potential to be a powerful tool in the analysis of population genetics and population structure as well as in the detection of gene flow.     

Species-Specific Detection of Vibrio anguillarum in Marine Aquaculture Environments by Selective Culture and DNA Hybridization

Methods for specific detection of Vibrio anguillarum in complex microbial communities within diverse marine aquaculture environments were evaluated. A system for the detection of culturable cells based on the combined use of a selective medium and a nonradioactively labeled oligodeoxynucleotide complementary to 16S rRNA was developed. Four hundred fourteen bacterial cultures were evaluated in order to assess the specificity of the method. When both the selective medium and the specific probe gave positive results, the cultures were always identified as V. anguillarum. The selectivity for colony hybridization was 1 V. anguillarum cell in 10,000 total bacterial cells in environmental samples. The utility of the method was also compared with detection by dot blot hybridization of either raw DNA purified from environmental samples or PCR-amplified DNA of 16S rRNA genes, using universal eubacterial primers. The post-PCR hybridization was more sensitive (8 x 10(sup2) cells) than direct hybridization of the whole purified DNA (10(sup6) cells). However, the selective medium-probe combined method was as sensitive as post-PCR hybridization, albeit more specific.     

Rapid detection and identification of a pathogen's DNA using Phi29 DNA polymerase

Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing as little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.     

Design and Performance of rRNA Targeted Oligonucleotide Probes for in Situ Detection and Phylogenetic Identification of Microorganisms Inhabiting Acid Mine Drainage Environments

At Iron Mountain, CA, there is an extreme occurrence of acid mine drainage (AMD). This is a result of past mining activity that has exposed a sulfide ore body to weathering and microbial activity. This study presents seven new oligonucleotide probes for the detection of microorganisms at this AMD site by fluorescent in situ hybridization. In the design of these probes we have accounted for a large body of 16S rRNA sequence data recently compiled by us. This was obtained by PCR and cloning directly from environmental DNA and was mostly represented by novel sequences. The probes were developed to include detection of novel and uncultivated organisms. This includes detection for the Thermoplasmales group, a new group of Leptospirillum, the genus Sulfobacillus, the Acidiphilium genus, Acidimicrobium and relatives, and for organisms within the delta Proteobacteria. These probes have been used to examine the abundance and distribution of organisms, including novel and uncultivated taxa, and to clarify their potential contributions to AMD production at the site. We anticipate that these probes will be useful tools for exploration of the microbiology of other natural acidic environments and bioleaching systems.     

Mining metagenomes for novel cellulase genes

Cellulases hydrolyze the β-1,4 linkages of cellulose and are widely used in food, brewing and wine, animal feed, textiles and laundry, and pulp and paper industries, especially for hydrolyzing cellulosic materials into sugars, which can be fermented to produce useful products such as ethanol. Metagenomics has become an alternative approach to conventional culture-dependent methods as it allows exhaustive mining of microbial genomes in their natural environments. This review covers the current state of research and challenges in mining novel cellulase genes from the metagenomes of various environments, and discusses the potential biotechnological applications of metagenome-derived cellulases.     

The distribution of dissolved DNA in an oligotrophic and a eutrophic river of Southwest Florida

The distribution of dissolved DNA concentrations and some microbial variables were compared in an oligo-mesotrophic river (the Crystal River) and a phosphate-rich eutrophic river (the Alafia River) in Southwest Florida over a 15 month period. Concentrations of phosphate and nitrate in the Alafia River averaged 135 and 18.2 times the respective phosphate and nitrate concentrations of the oligo-mesotrophic Crystal River. The seasonal average dissolved DNA concentration for the Alafia River exceeded that of the Crystal River by a factor of 1.8 (8.2 g 1^–1 compared to 4.6 g 1^–1, respectively). The greatest concentrations of dissolved DNA in the Alafia River were found in areas that contained the largest populations of phytoplankton and bacteria (a reservoir formed from an abandoned phosphate mining pit and two downstream stations near the mouth of the river). Differences in dissolved DNA concentrations between these environments and more pristine environments (i.e. all Crystal River Stations and upstream Alafia River stations) were of the same order of magnitude (1.8 to 2.2-fold) as the differences in bacterial abundance and activity, but considerably less than differences in phytoplankton abundance and activity between such environments. Seasonal variations in dissolved DNA concentrations in the Crystal River corresponded to seasonal variations in microbial populations, with minimal values in January and greater values in July. In the Alafia River, lowest concentrations for dissolved DNA occurred in July during the wet season, when seasonal flooding of area of leaf litter yielded high levels of dissolved organic carbon (DOC) which were low in dissolved DNA. These results suggest that: 1) in situ planktonic activity is a greater source of dissolved DNA than allochthonous or terrestrial sources of DOC; 2) factors that control the magnitude of heterotrophic bacterial populations are more likely to control dissolved DNA levels than factors regulating autotrophic population activity and abundance; 3) differences in dissolved DNA between eutrophic and oligo-mesotrophic environments are often much smaller than the differences in nutrient concentration between such environments.     

Rapid Detection and Quantification of Members of the Archaeal Community by Quantitative PCR Using Fluorogenic Probes

We describe a rapid, reproducible, and sensitive method for detection and quantification of archaea in naturally occurring microbial communities. A domain-specific PCR primer set and a domain-specific fluorogenic probe having strong and weak selectivity, respectively, for archaeal rRNA genes (rDNAs) were designed. A universal PCR primer set and a universal fluorogenic probe for both bacterial and archaeal rDNAs were also designed. Using these primers and probes, we demonstrated that detection and quantification of archaeal rDNAs in controlled microbial rDNA assemblages can be successfully achieved. The system which we designed was also able to detect and quantify archaeal rDNAs in DNA samples obtained not only from environments in which thermophilic archaea are abundant but also from environments in which methanogenic archaea are abundant. Our findings indicate that this method is applicable to culture-independent molecular analysis of microbial communities in various environments.     

Rapid detection and quantification of members of the archaeal community by quantitative PCR using fluorogenic probes

We describe a rapid, reproducible, and sensitive method for detection and quantification of archaea in naturally occurring microbial communities. A domain-specific PCR primer set and a domain-specific fluorogenic probe having strong and weak selectivity, respectively, for archaeal rRNA genes (rDNAs) were designed. A universal PCR primer set and a universal fluorogenic probe for both bacterial and archaeal rDNAs were also designed. Using these primers and probes, we demonstrated that detection and quantification of archaeal rDNAs in controlled microbial rDNA assemblages can be successfully achieved. The system which we designed was also able to detect and quantify archaeal rDNAs in DNA samples obtained not only from environments in which thermophilic archaea are abundant but also from environments in which methanogenic archaea are abundant. Our findings indicate that this method is applicable to culture-independent molecular analysis of microbial communities in various environments.     

Extraction and detection of baculoviral DNA from lake water, detritus and forest litter

AIMS: This paper describes a quick, reproducible, sensitive method for baculoviral DNA extraction, purification and detection from freshwater and forest litter environments. METHODS AND RESULTS: The extraction protocol utilizes enzymatic and chemical lysis and physical disruption. To assess the efficiency of the extraction and purification protocol, PCR was used to detect a 530 bp DNA fragment from the genome of a genetically-modified baculovirus, Choristoneura fumiferana NPVegt-/lacZ+. The detection limit of PCR amplification was routinely about 4.1 x 102 occlusion bodies (OBs) 450 microl-1 lake water. Template DNA from the detritus and forest litter samples required 100-fold dilutions before use in PCR reactions. The detection limits for detritus and forest litter samples were routinely about 7.41 x 103 and 2.08 x 104 OBs 0.5 g-1 dry weight, respectively. CONCLUSION: The DNA extraction and purification methodology is reproducible, sensitive and can be used in lieu of, or in conjunction with, insect bioassays. SIGNIFICANCE AND IMPACT OF THE STUDY: The DNA extraction and purification protocol described in this paper will facilitate risk assessment and ecological studies of both wild-type and genetically-modified baculoviruses.     

Application of polymerase chain reaction (PCR) and TaqMan PCR techniques to the detection and identification of Rhodococcus coprophilus in faecal samples.

Rhodococcus coprophilus, a natural inhabitant of herbivore faeces, has been suggested as a good indicator of animal (as opposed to human) faecal contamination of aquatic environments. However, conventional detection methods limit its use for this as they require up to 21 days to obtain a result. In this paper an optimised method for extracting R. coprophilus DNA from faecal samples is described. PCR and 5'-nuclease (TaqMan) PCR methods were developed to allow the detection and enumeration of R. coprophilus in faecal samples within 2-3 days. Both PCR methods targeted the 16S rRNA gene, producing an amplicon of 443 bp which was specific for R. coprophilus. Sixty cells were required to produce an amplification product by conventional PCR, while as little as one cell was required for the TaqMan PCR method. The latter approach gave a linear quantitative response over at least four log units with both bacterial cells and DNA. Successful amplification by PCR was achieved using DNA extracted from cow, sheep, horse and deer faeces but was negative for samples from humans, pig, possum, duck and rabbit. These PCR methods enhance the feasibility of using R. coprophilus to distinguish faecal pollution of farmed herbivores from human pollution.     

Sediment Contamination along Desert Wash Systems from Historic Mining Sites in a Hyperarid Region of Southern Nevada,USA

Abandoned mining sites in hyperarid environments are generally assumed to present an insignificant risk to water availability. This study investigated the impact abandoned mine sites in Southern Nevada can have on the wider environment. Southern Nevada is characterized with little precipitation and prolonged droughts. Precipitation in Southern Nevada is often in the form of short and intense events with the potential to mobilize and transport contaminated sediments down gradient. This work evaluated the movement of trace elements (Ag, As, Ba, Cd, Cr, Hg, Pb, and Se) and cyanide in surface sediments of three desert washes near the historic mining town of Nelson, a hyperarid region of Southern Nevada. Results indicate trace elements have been mobilized and transported down gradient from sources to areas not directly impacted by mining. Contaminants used in mining operations (Hg and CN^?) as well as those of geogenic nature migrated as far as 6000 m, providing evidence of their transport in hyperarid environments, contrary to the generally held belief. Although contaminants in this study are below levels that are environmentally significant, the findings show that transport is possible. This study demonstrates that large amounts of contaminant-laden sediments might be a significant threat in hyperarid environment and to areas down gradient from source materials.     

Genotoxic effects in wild rodents (Rattus rattus and Mus musculus) in an open coal mining area

Coal is a mixture of a variety of compounds containing mutagenic and carcinogenic polycyclic aromatic hydrocarbons. Exposure to coal is considered as an important non-cellular and cellular source of reactive oxygen species that can induce DNA damage. In addition, spontaneous combustion can occur in coal mining areas, further releasing compounds with detrimental effects on the environment. In this study the comet assay was used to investigate potential genotoxic effects of coal mining activities in peripheral blood cells of the wild rodents Rattus rattus and Mus musculus. The study was conducted in a coal mining area of the Municipio de Puerto Libertador, South West of the Departamento de Cordoba, Colombia. Animals from two areas in the coal mining zone and a control area located in the Municipio de Lorica were investigated. The results showed evidence that exposure to coal results in elevated primary DNA lesions in blood cells of rodents. Three different parameters for DNA damage were assessed, namely, DNA damage index, migration length and percentage damaged cells. All parameters showed statistically significantly higher values in mice and rats from the coal mining area in comparison to the animals from the control area. The parameter "DNA Damage Index" was found to be most sensitive and to best indicate a genotoxic hazard. Both species investigated were shown to be sensitive indicators of environmental genotoxicity caused by coal mining activities. In summary, our study constitutes the first investigation of potential genotoxic effects of open coal mining carried out in Puerto Libertador. The investigations provide a guide for measures to evaluate genotoxic hazards, thereby contributing to the development of appropriate measures and regulations for more careful operations during coal mining.     

Systematic evaluation of bias in microbial community profiles induced by whole genome amplification

Whole genome amplification methods facilitate the detection and characterization of microbial communities in low biomass environments. We examined the extent to which the actual community structure is reliably revealed and factors contributing to bias. One widely used [multiple displacement amplification (MDA)] and one new primer‐free method [primase‐based whole genome amplification (pWGA)] were compared using a polymerase chain reaction (PCR)‐based method as control. Pyrosequencing of an environmental sample and principal component analysis revealed that MDA impacted community profiles more strongly than pWGA and indicated that this related to species GC content, although an influence of DNA integrity could not be excluded. Subsequently, biases by species GC content, DNA integrity and fragment size were separately analysed using defined mixtures of DNA from various species. We found significantly less amplification of species with the highest GC content for MDA‐based templates and, to a lesser extent, for pWGA. DNA fragmentation also interfered severely: species with more fragmented DNA were less amplified with MDA and pWGA. pWGA was unable to amplify low molecular weight DNA (< 1.5 kb), whereas MDA was inefficient. We conclude that pWGA is the most promising method for characterization of microbial communities in low‐biomass environments and for currently planned astrobiological missions to Mars.     

Environmental DNA (eDNA): Powerful technique for biodiversity conservation

Environmental DNA metabarcoding is a non-invasive method for discovering and identifying rare and endangered species in a variety of ecosystems, including aquatic environments, based on the retrieval of genetic traces emitted into the environment by animals. Environmental (e) DNA research has grown in popularity over the last decade as a result of a rise in the number of studies that employ DNA taken from the environment, particularly in freshwater and marine ecosystems. In terms of detecting diversity patterns, we may claim that DNA retrieved from the environment (eDNA) is altering the game. For resource management in fisheries, information on species composition and biomass/abundance of commercially and noncommercially harvested species is critical. The eDNA is a truly non-invasive method that inflicts no damage on the species or habitats under study even during sampling, the eDNA technique never harms any ecosystems or threatened species. This novel molecular method never affects any endangered species or ecosystem during sampling. Environmental DNA analysis has become more widely accepted and is used in the detection of the presence and absence of aquatic macrofauna, such as freshwater and marine fish. This review study may aid researchers in better understanding the current state of eDNA technology. Despite the fact that various scientists have used eDNA to investigate the worldwide biodiversity of aquatic environments, no one in India is focusing on this new technology. We conclude that the eDNA technique has the potential to become a next-generation tool for biodiversity research and aquatic ecosystem conservation.     

Genotoxic effects in wild rodents (Rattus rattus and Mus musculus) in an open coal mining area

Coal is a mixture of a variety of compounds containing mutagenic and carcinogenic polycyclic aromatic hydrocarbons. Exposure to coal is considered as an important non-cellular and cellular source of reactive oxygen species that can induce DNA damage. In addition, spontaneous combustion can occur in coal mining areas, further releasing compounds with detrimental effects on the environment. In this study the comet assay was used to investigate potential genotoxic effects of coal mining activities in peripheral blood cells of the wild rodents Rattus rattus and Mus musculus. The study was conducted in a coal mining area of the Municipio de Puerto Libertador, South West of the Departamento de Cordoba, Colombia. Animals from two areas in the coal mining zone and a control area located in the Municipio de Lorica were investigated. The results showed evidence that exposure to coal results in elevated primary DNA lesions in blood cells of rodents. Three different parameters for DNA damage were assessed, namely, DNA damage index, migration length and percentage damaged cells. All parameters showed statistically significantly higher values in mice and rats from the coal mining area in comparison to the animals from the control area. The parameter “DNA Damage Index” was found to be most sensitive and to best indicate a genotoxic hazard. Both species investigated were shown to be sensitive indicators of environmental genotoxicity caused by coal mining activities. In summary, our study constitutes the first investigation of potential genotoxic effects of open coal mining carried out in Puerto Libertador. The investigations provide a guide for measures to evaluate genotoxic hazards, thereby contributing to the development of appropriate measures and regulations for more careful operations during coal mining.