Detection of Naegleria fowleri cysts in environmental samples by using a DNA probe

Abstract In this study we tried to detect DNA Naegleria fowleri in artificially contaminated environmental samples, with or without sediments, containing 10⁴ cysts of this pathogenic amoeba. We used two assays to extract DNA from samples: first, direct DNA extraction, which gave positive results only for water samples without sediment; second, DNA extraction after sample incubation on agar plates, which allowed us to remove amoeba growing out of the sediments, and which gave positive results for all samples, even those initially with sediments (5, 500 or 500 mg). Thus, this molecular identification appears as a powerful tool to investigate N. fowleri growth in environmental samples.     

Detection of a diverse marine fish fauna using environmental DNA from seawater samples

Marine ecosystems worldwide are under threat with many fish species and populations suffering from human over-exploitation. This is greatly impacting global biodiversity, economy and human health. Intriguingly, marine fish are largely surveyed using selective and invasive methods, which are mostly limited to commercial species, and restricted to particular areas with favourable conditions. Furthermore, misidentification of species represents a major problem. Here, we investigate the potential of using metabarcoding of environmental DNA (eDNA) obtained directly from seawater samples to account for marine fish biodiversity. This eDNA approach has recently been used successfully in freshwater environments, but never in marine settings. We isolate eDNA from ½-litre seawater samples collected in a temperate marine ecosystem in Denmark. Using next-generation DNA sequencing of PCR amplicons, we obtain eDNA from 15 different fish species, including both important consumption species, as well as species rarely or never recorded by conventional monitoring. We also detect eDNA from a rare vagrant species in the area; European pilchard (Sardina pilchardus). Additionally, we detect four bird species. Records in national databases confirmed the occurrence of all detected species. To investigate the efficiency of the eDNA approach, we compared its performance with 9 methods conventionally used in marine fish surveys. Promisingly, eDNA covered the fish diversity better than or equal to any of the applied conventional methods. Our study demonstrates that even small samples of seawater contain eDNA from a wide range of local fish species. Finally, in order to examine the potential dispersal of eDNA in oceans, we performed an experiment addressing eDNA degradation in seawater, which shows that even small (100-bp) eDNA fragments degrades beyond detectability within days.Although further studies are needed to validate the eDNA approach in varying environmental conditions, our findings provide a strong proof-of-concept with great perspectives for future monitoring of marine biodiversity and resources.     

Detection of Mycobacterium leprae DNA in clinical and environmental samples using serological analysis and PCR

Molecular Biology Reports - Leprosy is a chronic infectious disease caused by Mycobacterium leprae and persists as a serious public health problem in Brazil. This microorganism is inculturable,...     

Detection of PCR amplicons from bacterial pathogens using microsphere agglutination

For rapid and inexpensive detection of polymerase chain reaction (PCR) amplicons, a novel microsphere agglutination assay has been developed. PCR is carried out using biotinylated forward and reverse primers, and the amplified DNA fragments are able to agglutinate streptavidin-coated microspheres (5.7 microm in diameter). Purification of PCR amplicons is unnecessary when initial primer concentrations are 250 nM. Agglutination can be identified visually within 2 min without any additional equipment or reagents. Using listeriolysin (lisA)-specific biotinylated primers, we have successfully detected and identified Listeria monocytogenes lisA+ cells among Salmonella typhimurium, Staphylococcus aureus, Campylobacter jejuni and Escherichia coli O157:H7 cells. The simplicity of this protocol considerably reduces the time and cost of diagnostic PCR experiments. This procedure is potentially useful for various studies and field applications.     

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.     

Hybridization of genomic DNA to microarrays: a challenge for the analysis of environmental samples

The use of DNA microarrays for detection and identification of bacteria and genes of interest from various environments (e.g. soil, sediment, water column...) is a major challenge for microbiologists working on functional diversity. So far, most of the genomic methods that have been described rely on the use of taxonomic markers (such as 16S rRNA) that can be easily amplified by PCR prior to hybridization on microarrays. However, taxonomical markers are not always informative on the functions present in these bacteria. Moreover, genes for which sequence database is limited or that lack any conserved regions will be difficult to amplify and thus to detect in unknown samples. Furthermore, PCR amplification often introduces biases that lead to inaccurate analysis of microbial communities. An alternative solution to overcome these strong limitations is to use genomic DNA (gDNA) as target for hybridisation, without prior PCR amplification. Though hybridization of gDNA is already used for comparative genome hybridization or sequencing by hybridization, yet to the high cost of tiling strategies and important data filtering, its adaptation for use in environmental research poses great challenges in terms of specificity, sensitivity and reproducibility of hybridization. Considering the very faint number of publications that have described hybridization of gDNA to microarrays for environmental applications, we confront in this review the different approaches that have been developed so far, and propose alternative strategies that may contribute to improve the development of microarrays for studying the microbial genetic structure and composition of samples of high environmental and ecological value.     

Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays

We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer. PCR products (ca. 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55 degrees C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 x 10(6) genomes; 5 ng) or by the addition of up to 500 ng of fish DNA. Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens.     

PNA microarrays for hybridisation of unlabelled DNA samples

Several strategies have been developed for the production of peptide nucleic acid (PNA) microarrays by parallel probe synthesis and selective coupling of full-length molecules. Such microarrays were used for direct detection of the hybridisation of unlabelled DNA by time-of-flight secondary ion mass spectrometry. PNAs were synthesised by an automated process on filter-bottom microtitre plates. The resulting molecules were released from the solid support and attached without any purification to microarray surfaces via the terminal amino group itself or via modifications, which had been chemically introduced during synthesis. Thus, only full-length PNA oligomers were attached whereas truncated molecules, produced during synthesis because of incomplete condensation reactions, did not bind. Different surface chemistries and fitting modifications of the PNA terminus were tested. For an examination of coupling selectivity, bound PNAs were cleaved off microarray surfaces and analysed by MALDI-TOF mass spectrometry. Additionally, hybridisation experiments were performed to compare the attachment chemistries, with fully acetylated PNAs spotted as controls. Upon hybridisation of unlabelled DNA to such microarrays, binding events could be detected by visualisation of phosphates, which are an integral part of nucleic acids but missing entirely in PNA probes. Overall best results in terms of selectivity and sensitivity were obtained with thiol-modified PNAs on maleimide surfaces.     

Detection of bacteria in environmental samples by direct PCR without DNA extraction

Cultured cells and environmental samples were used directly in PCRs without the isolation of DNA. Serial dilution was used to eliminate the inhibitory effect of materials in natural samples. Primers specific for pmoA, which encodes a subunit of the particulate methane monooxygenase, were used to detect and quantify methanotrophic bacteria by direct most probable number PCR. Phototrophic bacteria were detected in environmental samples by direct PCR with primers specific for pufM, and members of the bacterial domain were detected with primers for 16S rDNA. Direct PCR provides a rapid, simple, and sensitive methodfor detecting and quantifying bacteria in environmental samples. Detection of methanotrophic bacteria can be applied to monitoring bioremediation.     

基因芯片技术在检测肠道致病菌方面的应用

基因芯片技术具有高通量、自动化、快速检测等特点,因此被广泛地应用于各种研究领域,如细菌分子流行病学、细菌基因鉴定、致病分子机理、基因突变及多态性分析、表达谱分析、DNA测序和药物筛选等。现介绍基因芯片检测肠道致病菌方面的国外研究进展,基因芯片应用于检测肠道致病菌的3个方面:结合多重PCR对致病菌的毒力因子或者特异性基因进行鉴定;直接检测细菌的DNA或者RNA;以致病细菌核糖体RNA作为检测的靶基因同时检测多种肠道致病菌。由于其检测的高效率,该技术要优于其他分子生物学检测方法。基因芯片技术在肠道致病菌检测中有着巨大的应用价值,具有广阔的应用前景。     

Detection and identification of poliovirus in environmental samples using nucleic acid hybridization

A procedure was developed to effectively extract viral RNA from poliovirus tissue-culture lysates while eliminating the hybridization background associated with tissue cultures uninfected with poliovirus. Poliovirus cDNA cloned into a pUC vector was used as probe. Both the recombinant plasmids and the cDNA showed great specificity towards poliovirus. However, both probes hybridized with the single-stranded DNA coliphage phi X174. Tissue culture was found to be an effective method to increase the number of viruses found in environmental samples to a level detectable by hybridization procedures, whereas direct hybridization of RNA from unamplified and highly concentrated raw wastewater showed poor hybridization signals. The specificity and sensitivity of the hybridization procedure developed during these studies indicate that this method may be best suited for the identification rather than the detection of viruses isolated from environmental samples.     

Detection of foodborne pathogens using DNA probes and a dipstick format

The detection of foodborne microorganisms has traditionally been done using microbiologically based methods. Such “gold standard” methods are generally reliable but have the disadvantages of being labor intensive, subjective, and time consuming. Over the last several years, the development of DNA probe-based methods has simplified the methods used to detect organisms such asSalmonella, Listeria, andE. coli by targeting the unique DNA or RNA sequences of these organisms using DNA probes and nonradioactive detection.     

Comparing whole genomes using DNA microarrays

The rapid accumulation of complete genomic sequences offers the opportunity to carry out an analysis of inter- and intra-individual genome variation within a species on a routine basis. Sequencing whole genomes requires resources that are currently beyond those of a single laboratory and therefore it is not a practical approach for resequencing hundreds of individual genomes. DNA microarrays present an alternative way to study differences between closely related genomes. Advances in microarray-based approaches have enabled the main forms of genomic variation (amplifications, deletions, insertions, rearrangements and base-pair changes) to be detected using techniques that are readily performed in individual laboratories using simple experimental approaches.     

Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50 degrees C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10(2) to 10(3) CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.     

Detection of bacterial pathogens in municipal wastewater using an oligonucleotide microarray and real-time quantitative PCR

As a first step toward building a comprehensive microarray, two low density DNA microarrays were constructed and evaluated for the accurate detection of wastewater pathogens. The first one involved the direct hybridization of wastewater microbial genomic DNA to the functional gene probes while the second involved PCR amplification of 23S ribosomal DNA. The genomic DNA microarray employed 10 functional genes as detection targets. Sensitivity of the microarray was determined to be approximately 1.0 microg of Esherichia coli genomic DNA, or 2 x 10(8) copies of the target gene, and only E. coli DNA was detected with the microarray assay using municipal raw sewage. Sensitivity of the microarray was enhanced approximately by 6 orders of magnitude when the target 23S rRNA gene sequences were PCR amplified with a novel universal primer set and allowed hybridization to 24 species-specific oligonucleotide probes. The minimum detection limit was estimated to be about 100 fg of E. coli genomic DNA or 1.4 x 10(2) copies of the 23S rRNA gene. The PCR amplified DNA microarray successfully detected multiple bacterial pathogens in wastewater. As a parallel study to verify efficiency of the DNA microarray, a real-time quantitative PCR assay was also developed based on the fluorescent TaqMan probes (Applied Biosystems).     

Detection of invasive freshwater fish species using environmental DNA survey

Invasive species are one of the most significant problem in freshwater ecosystems. Most common non-native freshwater species in Turkish freshwater fish fauna are Prussian Carp (Carassius gibelio), North African Catfish (Clarias gariepinus), Nile Tilapia (Oreochromis niloticus) and Topmouth Gudgeon (Pseudorasbora parva).Recent studies showed that environmental DNA could be used to detect target species inhabiting the ecosystem with higher precision and less effort compared to traditional field surveys. In this study, eDNA approach was used to investigate non-native freshwater fish species from fifteen different locations of Upper Sakarya Basin. eDNA was successfully extracted from the water samples of locations where the species were visually observed. Mean amplification rate of eDNA was calculated as 77.03%.This study is the first environmental DNA study used in detection of four of the most common invasive freshwater fish species. Results clearly indicating that eDNA surveys could be used as an important molecular tool to monitor invasive fish species in freshwater ecosystems.     

Evaluation of soil bacterial biomass using environmental DNA extracted by slow-stirring method

A simple and rapid method (slow-stirring method) for extracting environmental DNA (eDNA) from soils was constructed by physical mild stirring with chemical treatment. eDNA was extracted efficiently with minimal damage from various kinds of soil. The amount of eDNA and soil bacterial biomass showed a linear proportional relation [Y=(1.70×10⁸)X, r ²=0.96], indicating that bacterial biomass could be evaluated by quantifying levels of eDNA. Consequently, the average bacterial biomass in an agricultural field was calculated as 5.95×10⁹ cells/g sample, approximately 10–100 times higher than that in non- and oil-polluted fields.     

DNA-based monitoring of total bacterial community structure in environmental samples

Determining the structure of bacterial communities and their response to stimuli is key to understanding community function and the interactions that occur between microorganisms and the environment. However, bacterial communities often comprise complex assemblages of large numbers of different bacterial populations. An approach is presented which allows bacterial community structure to be determined by fractionation of the complex mixture of total bacterial community DNA using the DNA-binding dye bisbenzimidazole which imposes G + C-dependent changes in the buoyant density of DNA. Bacterial community structure presented as percentage of total DNA vs. percentage G + C content of DNA is an indication of the relative abundance of phylogenetic groups of bacteria. Changes in the composition of a soil bacterial community in response to perturbations in the form of carbon amendment and altered water status were monitored.