Microdevice‐based solid‐phase polymerase chain reaction for rapid detection of pathogenic microorganisms

We demonstrate the integration of DNA amplification and detection functionalities developed on a lab‐on‐a‐chip microdevice utilizing solid‐phase polymerase chain reaction (SP‐PCR) for point‐of‐need (PON) DNA analyses. First, the polycarbonate microdevice was fabricated by thermal bonding to contain microchambers as reservoirs for performing SP‐PCR. Next, the microchambers were subsequently modified with polyethyleneimine and glutaraldehyde for immobilizing amine‐modified forward primers. During SP‐PCR, the immobilized forward primers and freely diffusing fluorescence‐labeled reverse primers cooperated to generate target amplicons, which remained covalently attached to the microchambers for the fluorescence detection. The SP‐PCR microdevice was used for the direct identifications of two widely detected foodborne pathogens, namely Salmonella spp. and Staphylococcus aureus, and an alga causing harmful algal blooms annually in South Korea, Cochlodinium polykrikoides. The SP‐PCR microdevice would be versatilely applied in PON testing as a universal platform for the fast identification of foodborne pathogens and environmentally threatening biogenic targets.     

Human papillomavirus detection by non isotopic in situ hybridization, in situ hybridization with signal amplification and in situ polymerase chain reaction

Classical in situ hybridization (ISH) with biotinylated probes makes it possible to detect and localize human papillomavirus (HPV) nucleic acid sequences in cytological and histological materials. This method is however of limited value in the detection of a few copies of the virus. Moreover the specificity of such a technique is not always convincing when ISH signals are small and/or of low intensity. Recently, much attention has been focused on the utility of the in vitro polymerase chain reaction (PCR) and especially on PCR-single strand conformation polymorphism (SSCP) to amplify small amounts of viral DNA with accurate hybrid specificity. But the latter method requires nucleic acid extraction and tissue destruction. Thus, correlation between the PCR results and histological findings is not possible. Hence, the aim of our current study was to apply to HeLa cells and cervical formalin-fixed and paraffin-embedded biopsies, a novel procedure of ISH signal amplification, the catalyzed signal amplification (CSA). Such a procedure is based on the deposition of streptavidin-horseradish peroxidase catalyzing the deposition of biotinylated tyramide molecules on the location of the probed target. The biotin accumulation is then detected with streptavidin peroxidase and diaminobenzidine. The results were compared with those obtained by direct and indirect in situ PCR. The catalysed signal amplification successfully increased the sensitivity and efficiency of ISH for the detection of rare sequences in HPV infected cells and histological materials. Such a method was found simpler and faster than in situ PCR and tissue morphology was better preserved.     

Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms

As a technique allowing simultaneous visualization, identification, enumeration and localization of individual microbial cells, fluorescence in situ hybridization (FISH) is useful for many applications in all fields of microbiology. FISH not only allows the detection of culturable microorganisms, but also of yet-to-be cultured (so-called unculturable) organisms, and can therefore help in understanding complex microbial communities. In this review, methodological aspects, as well as problems and pitfalls of FISH are discussed in an examination of past, present and future applications.     

In situ detection of starch-hydrolyzing microorganisms in activated sludge

Polysaccharides constitute a significant part of the organic matter in domestic wastewater and their hydrolysis plays an important role in their transformation and nutrient removal in activated sludge wastewater treatment plants. However, there is no information available about the identity, ecophysiology, and abundance of starch-hydrolyzing organisms (SHOs) in these plants. In this study, fluorescence in situ enzyme staining with BODIPY fluorescein-labeled starch was applied and optimized to label SHOs expressing alpha-amylase in activated sludge plants. Fluorescence on the surface of bacteria expressing alpha-amylase activity was clearly visualized. In 11 full-scale nutrient-removing wastewater treatment plants examined, the morphotypes of the dominant SHOs were always cocci in clusters of tetrads, short rods in clusters, and some filamentous organisms. The SHOs were identified by combining in situ enzyme staining and FISH using a range of available oligonucleotide probes. All the SHOs observed were Actinobacteria, and most had the phenotype of polyphosphate-accumulating organisms closely related to the genus Tetrasphaera in the family Intrasporangiaceae. The SHOs were present in most of the wastewater treatment plants examined and comprised, in total, up to 11% of bacterial biovolume and thus formed an important part of the microbial communities.     

In situ detection of protein-hydrolysing microorganisms in activated sludge

Protein hydrolysis plays an important role in the transformation of organic matter in activated sludge wastewater treatment plants, but no information is currently available regarding the identity and ecophysiology of protein-hydrolysing organisms (PHOs). In this study, fluorescence in situ enzyme staining with casein and bovine serum albumin conjugated with BODIPY dye was applied and optimized to label PHOs in activated sludge plants. A strong fluorescent labeling of the surface of microorganisms expressing protease activity was achieved. Metabolic inhibitors were applied to inhibit the metabolic activity to prevent uptake of the fluorescent hydrolysates by oligopeptide-consuming bacteria. In five full-scale, nutrient-removing activated sludge plants examined, the dominant PHOs were always different morphotypes of filamentous bacteria and the epiflora attached to many of these. The PHOs were identified by FISH using a range of available oligonucleotide probes. The filamentous PHOs belonged to the candidate phylum TM7, the phylum Chloroflexi and the class Betaproteobacteria. In total they comprised 1-5% of the bacterial biovolume. Most of the epiflora-PHOs hybridized with probe SAP-309 targeting Saprospiraceae in the phylum Bacteroidetes and accounted for 8-12% of the total bacterial biovolume in most plants and were thus an important and dominant part of the microbial communities.     

Applications of real-time polymerase chain reaction for quantification of microorganisms in environmental samples

Due to the advanced development of fluorogenic chemistry, quantitative real-time polymerase chain reaction (qRT-PCR) has become an emerging technique for the detection and quantification of microorganisms in the environment. Compared with the conventional hybridization- and PCR-based techniques, qRT-PCR not only has better sensitivity and reproducibility, but it is also quicker to perform and has a minimum risk of amplicon carryover contamination. This article reviews the principle of this emerging technique, its detection reagents, target DNAs, quantification procedures, and affecting factors. The applications of qRT-PCR for the quantification of microorganisms in the environment are also summarized.     

The detection of transgenic animals using a polymerase chain reaction in situ

The technique for detecting both foreign and host specific DNA sequences inside nuclei and chromosomes of single cells of transgenic mice with the help of polymerase chain reaction (PCR) in situ is described. The mouse preimplantation and postimplantation embryonic and adult cells were studied. The methodology is described in detail with particular attention to the optimization of composition of reaction mixture, kind of fixation and preliminary denaturation of target DNA. The reaction takes only several hours and needs no sophisticated equipment.     

Cloth-based hybridization array system for detection of transgenic soy and corn by multiplex polymerase chain reaction

A cloth-based hybridization array system (CHAS) was developed for detection of amplicons generated in a multiplex PCR targeting transgenic 35S and NOS sequences, as well as corn invertase and soy lectin genes. The CHAS provided confirmation of each amplicon on the basis of hybridization with specific capture probes, and enabled ease of discrimination of the multiplex PCR products by visualization of the amplicons on the array.     

In situ polymerase chain reaction and cycling primed in situ amplification: improvements and adaptations

 Ethanol fixation combined with microwave pretreatment allows rapid and simple detection of signals produced by cycling primed in situ (PRINS) amplification, which uses a single primer, and in situ polymerase chain reaction (ISPCR) in intact cells. After thermal cycling, signals remain as discrete subnuclear spots in the region of amplification and are clearly distinguishable from non-specific background labelling. These methods are applicable to routine blood smears, even after Giemsa staining or immunocytochemistry, and cellular morphology is retained. Chromosome enumeration by cycling PRINS is demonstrated using primers for repeat DNA sequences, whilst single copy sequence detection is demonstrated using bcl-2, CFTR and chromosome 21 specific primer pairs in ISPCR. We show that ethanol fixation supports efficient extension of cycling PRINS products to approximately 550 bp using up to 70 rounds of thermal cycling. Accepted: 15 February 1999     

Catalyzed reporter deposition-fluorescent in situ hybridization (CARD-FISH) detection of Dehalococcoides

Members of the genus Dehalococcoides are well-known for their capacity to reductively dechlorinate chlorinated organic pollutants. The availability of quantitative and sensitive detection methods is of major interest for research on the ecology of those environmentally important micro-organisms. In this paper we describe the development of a Catalyzed Reporter Deposition-Fluorescent In Situ Hybridization (CARD-FISH) for detection of Dehalococcoides cells in enrichment cultures using two oligonucleotide sequences which target two different lineages of Dehalococcoides as probes. Both sequences were previously applied in conventional FISH as probes. Conjugation of the probe to horseradish peroxidase (HRP) did not change the specificity of the probes and bright fluorescent signals were obtained. Despite the use of higher concentrations of probe and the application of longer exposure times in the conventional FISH procedure, CARD-FISH resulted in more intense signals. The CARD-FISH method was applied to a vinyl chloride (VC)-reductively-dechlorinating enrichment culture. Only the probe targeting the CBDB1 lineage of Dehalococcoides reacted with the sample which was in agreement with previous nucleic acid based analysis. The culture consisted of 51%+/-8% of Dehalococcoides cells. Furthermore, the CARD-FISH probes for detecting Dehalococcoides were combined with FISH probes for simultaneous detection of either Bacteria or Archaea which should allow rapid insight into the relative dynamics of the different members of dechlorinating communities as a response to environmental changes. Overall, CARD-FISH proved to be a rapid, reliable and convenient method to detect and quantify Dehalococcoides cells.     

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

Targeted species‐specific and community‐wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R² = 0.81, p < .001, biofouling R² = 0.68, p < .001); however, qPCR copy numbers were on average 125‐fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data.     

Linking environmental processes to the in situ functioning of microorganisms by high‐resolution secondary ion mass spectrometry (NanoSIMS) and scanning transmission X‐ray microscopy (STXM)

Environmental microbiology research increasingly focuses on the single microbial cell as the defining entity that drives environmental processes. The interactions of individual microbial cells with each other, the environment and with higher organisms shape microbial communities and control the functioning of whole ecosystems. A single‐cell view of microorganisms in their natural environment requires analytical tools that measure both cell function and chemical speciation at the submicrometre scale. Here we review the technical capabilities and limitations of high‐resolution secondary ion mass spectrometry (NanoSIMS) and scanning transmission (soft) X‐ray microscopy (STXM) and give examples of their applications. Whereas NanoSIMS can be combined with isotope‐labelling, thereby localizing the distribution of cellular activities (e.g. carbon/nitrogen fixation/turnover), STXM provides information on the location and chemical speciation of metabolites and products of redox reactions. We propose the combined use of both techniques and discuss the technical challenges of their joint application. Both techniques have the potential to enhance our understanding of cellular mechanisms and activities that contribute to microbially mediated processes, such as the biogeochemical cycling of elements, the transformation of contaminants and the precipitation of mineral phases.     

PrimRglo: a multiplexable quantitative real-time polymerase chain reaction system for nucleic acid detection

We report the development of a new real-time polymerase chain reaction (PCR) detection system that uses oligonucleotide "tagged" PCR primers, a fluorophore-labeled "universal" detection oligonucleotides, and a complementary quenching oligonucleotide. The fluorescence signal decreases as PCR product accumulates due to the increase in detection/quencher hybrid formation as the tagged primer is consumed. We use plasmids containing the influenza A matrix gene and the porA and ctrA genes of Neisseria meningitidis as targets for developing the system. Cycle threshold (Ct) values were generated, and the sensitivity of the new system (dubbed "PrimRglo") compared favorably with the commonly used SYBR green and Taqman detection systems and, unlike the latter system, does not require the design of a new dual-labeled detection oligonucleotide for each new target sequence.     

Improved DNA‐FISH for cytometric detection of Candida spp

Aims: We developed improved methods for DNA‐based fluorescence in situ hybridization (FISH) for rapid detection of Candida spp. and Candida albicans via flow cytometry. Methods and Results: Two previously reported C. albicans‐targeted DNA probes were evaluated against whole cells of C. albicans and related Candida species using a rapid, high‐temperature hybridization protocol. One probe (CalB2208) was shown for the first time to be suitable as a FISH probe. Although cell labelling for both probes was relatively bright, we were able to substantially improve our results by altering fixation and hybridization conditions. For fixation, a 60 : 40 mixture of 10% buffered formalin and ethanol was most effective. Probe intensity was improved as much as ten‐fold through the use of unlabelled helper probes, and buffer containing 0·9 mol l^?1 NaCl plus 10% formamide yielded the best hybridizations for both probe/helper cocktails. Although optimal labelling occurred with longer hybridizations, we found that C. albicans could be completely differentiated from the nontarget yeast Rhodotorula glutinis after only 15 min using the brightest probe (Calb‐1249) and that a formal washing step was not required. Specificities of probe/helper cocktails under optimal conditions were determined using a panel of target and nontarget cell types, including four strains of Candida dubliniensis. Calb‐1249 cross‐reacted slightly with Candida parapsilosis and strongly with both Candida tropicalis and C. dubliniensis. In contrast, we found that CalB2208 was exclusive for C. albicans. The molecular basis of this specificity was confirmed by DNA sequencing. Conclusions: We describe DNA probe‐based approaches for rapid and bright labelling of Candida spp. and for specific labelling of C. albicans without cross‐reaction with C. dubliniensis. Our work improves upon previously described methods. Significance and Impact of the Study: The methods described here for rapid FISH‐based detection of Candida spp. may have applications in both clinical and food microbiology.     

DNA‐based chemiluminescent nanoprobes for highly sensitive and selective detection of mercury(II) ion

A simple and sensitive DNA‐stablized gold nanoparticle (AuNP)‐based chemiluminescent (CL) probe for detecting mercury ion (Hg²⁺) in aqueous solution has been developed. The CL strategy relies upon the catalytic activity of unmodified AuNPs on the luminol–H₂O₂ CL reaction, and the interaction of unmodified AuNPs with DNA. The unmodified AuNPs can effectively differentiate unstructured and folded DNA. The DNA desorbs from AuNPs in the presence of Hg²⁺, leading to the increase in CL signal. By rationally varying the number of thymine in single‐strand oligonucleotides, the detection range could be tuned. Employing single‐strand oligonucleotides with 14 thymine in the detecting system, a sensitive linear range for Hg²⁺ ions from 5.0 × 10^–10 to 1.0 × 10^–7 mol/L and a detection limit of 2.1 × 10^–10 mol/L are obtained. Changing the number of thymine to 10 and 6, it leads to a narrow detection range but a high sensitivity. Besides, DNA‐based CL nanoprobes exhibit a remarkable selectivity for Hg²⁺ ions over a variety of competing metal ions. Copyright © 2012 John Wiley & Sons, Ltd.