Development and application of an oligonucleotide microarray for the detection of food-borne bacterial pathogens

The rapid and accurate detection and identification of food-borne pathogenic bacteria is critical for food safety. In this paper, we describe a rapid (<4 h) high-throughput detection and identification system that uses universal polymerase chain reaction (PCR) primers to amplify a variable region of bacterial the 16S rRNA gene, followed by reverse hybridization of the products to species-specific oligonucleotide probes on a chip. This procedure was successful in discriminating 204 strains of bacteria from pure culture belonging to 13 genera of bacteria. When this method was applied directly to 115 strains of bacteria isolated from foods, 112/115 (97.4%) were correctly identified; two strains were indistinguishable due to weak signal, while one failed to produce a PCR product. The array was used to detect and successfully identify two strains of bacteria from food poisoning outbreak samples, giving results through hybridization that were identical to those obtained by traditional methods. The sensitivity of the microarray assay was 10² CFU of bacteria. Thus, the oligonucleotide microarray is a powerful tool for the detection and identification of pathogens from foods. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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).     

Hybridization kinetics analysis of an oligonucleotide microarray for microRNA detection

MicroRNA (miRNA) microarrays have been successfully used for profiling miRNA expression in many physiological processes such as development, differentiation, oncogenesis, and other disease processes. Detecting miRNA by miRNA microarray is actually based on nucleic acid hybridization between target molecules and their corresponding complementary probes. Due to the small size and high degree of similarity among miRNA sequences, the hybridization condition must be carefully optimized to get specific and reliable signals. Previously, we reported a microarray platform to detect miRNA expression. In this study, we evaluated the sensitivity and specificity of our microarray platform. After systematic analysis, we determined an optimized hybridization condition with high sensitivity and specificity for miRNA detection. Our results would be helpful for other hybridization-based miRNA detection methods, such as northern blot and nuclease protection assay.     

Label-free impedance detection of oligonucleotide hybridisation on interdigitated ultramicroelectrodes using electrochemical redox probes

The direct detection of oligodeoxynucleotide (ODN) hybridisation using electrochemical impedance spectroscopy was made on interdigitated array (IDA) gold (Au) ultramicroelectrodes manufactured by silicon technology. The immobilisation of single stranded ODNs (ssODNs) was accomplished by self-assembling of thiol-modified ODNs onto an Au-electrode surface. Faradaic impedance was measured in the presence of K(3)[Fe(CN)(6)]. Double strand formation was identified by a decrease of approximately 50% in impedance in the low frequency region in the presence of K(3)[Fe(CN)(6)], compared to the spectrum of single stranded ODN. The frequency dependent diffusion of Fe(CN)(6)(3-) ions through defects in the ODN monolayer determines the impedance of Au-ssODN surface. The influence of DNA intercalator methylene blue on the impedance of both, single and double strands, was examined along with K(3)[Fe(CN)(6)] and confirmed by cyclic voltammetry. The layer densities and the hybridisation have been further corroborated by chronoamperometric redox recycling of para-aminophenol (p-AP) in ELISA like experiments. It can be concluded, that a performed impedance spectroscopy did not change the layer density. The impedance spectroscopy at ultramicroelectrodes combined with faradaic redox reactions enhances the impedimetric detection of DNA hybridisation on IDA platforms.     

Development of an oligonucleotide microarray for the detection and monitoring of marine dinoflagellates

Harmful Algal Blooms (HABs), mainly caused by dinoflagellates and diatoms, have great economic and sanitary implications. An important contribution for the comprehension of HAB phenomena and for the identification of risks related to toxic algal species is given by the monitoring programs. In the microscopy-based monitoring methods, harmful species are distinguished through their morphological characteristics. This can be time consuming and requires great taxonomic expertise due to the existence of morphologically close-related species. The high throughput, automation possibility and specificity of microarray-based detection assay, makes this technology very promising for qualitative detection of HAB species. In this study, an oligonucleotide microarray targeted to the ITS1-5.8S-ITS2 rDNA region of nine toxic dinoflagellate species/clades was designed and evaluated. Two probes (45-47 nucleotides in length) were designed for each species/clade to reduce the potential for false positives. The specificity and sensitivity of the probes were evaluated with ITS1-5.8S-ITS2 PCR amplicons obtained from 20 dinoflagellates cultured strains. Cross hybridization experiments confirmed the probe specificity; moreover, the assay showed a good sensitivity, allowing the detection of up to 2 ng of labeled PCR product. The applicability of the assay with field samples was demonstrated using net concentrated seawater samples, un-spiked or spiked with known amounts of cultured cells. Despite the general application of microarray technology for harmful algae detection is not new, a peculiar group of target species/clades has been included in this new-format assay. Moreover, novelties regarding mainly the probes and the target rDNA region have allowed sensitivity improvements in comparison to previously published studies.     

Identification of human pathogens isolated from blood using microarray hybridisation and signal pattern recognition

Background  

Pathogen identification in clinical routine is based on the cultivation of microbes with subsequent morphological and physiological characterisation lasting at least 24 hours. However, early and accurate identification is a crucial requisite for fast and optimally targeted antimicrobial treatment. Molecular biology based techniques allow fast identification, however discrimination of very closely related species remains still difficult.     

Beneficial effects of probiotics in upper respiratory tract infections and their mechanical actions to antagonize pathogens

Probiotics are live micro‐organisms with beneficial effects on human health, which have the ability to counteract infections at different locations of the body. Clinical trials have shown that probiotics can be used as preventive and therapeutic agents in upper respiratory tract infections (URTIs) and otitis. Their mechanical properties allow them to aggregate and to compete with pathogens for nutrients, space and attachment to host cells. Consequently, they can directly antagonize pathogens and thus exert beneficial effects without directly affecting the metabolism of the host. An overview of the probiotics with such traits, tested up to date in clinical trials for the prevention or treatment of URTIs and otitis, is presented in this review. Their mechanical properties in the respiratory tract as well as at other locations are also cited. Species with interesting in vitro properties towards pharyngeal cells or against common respiratory pathogens have also been included. The potential safety risks of the cited species are then discussed. This review could be of help in the screening of probiotic strains with specific mechanical properties susceptible to have positive effects in clinical trials against URTIs.     

An oligonucleotide microarray for detection and discrimination of orthopoxviruses based on oligonucleotide sequences of two viral genes

An oligonucleotide microarray for detection and identification of orthopoxviruses was developed. Genus specific and orthopoxvirus species-specific regions of the genes encoding chemokine binding and alpha/beta-interferon binding proteins were used as a target. The developed microarray allows the variola, monkeypox, cowpox, vaccinia, camel-pox and ectromelia (mousepox) viruses to be distinguished with a high degree of reliability.     

Spatial-temporal variability in diazotroph assemblages in Chesapeake Bay using an oligonucleotide nifH microarray

The distribution of nitrogen-fixing microorganisms in the Chesapeake Bay was investigated using fingerprints from a nifH microarray comprised of 706 60-mer oligonucleotide nifH probes representing cultivated organisms and environmental clones from different nifH clusters. Diverse nifH targets, amplified from samples using degenerate nifH primers, were detected in water column and sediment samples collected in April and October, 2001-2002. Total nifH richness and diversity (Simpson's and Shannon indices) were highest at the most riverine, oligohaline North Bay station. In most samples, the highest diversity was in nifH Cluster 3, which includes many anaerobes, while Cluster 1 (alpha-, beta- gamma- Proteobacteria, Cyanobacteria) targets had the greatest microarray signal intensities. In a multidimensional scaling analysis, deep water communities from April and October were similar within each of the sampling sites, while the surface communities had more variability. Diazotroph communities in the water column in the North Bay were distinct from the Mid- and South Bay communities, and there was a gradual change in sediment diazotroph assemblages from the North to the South Bay. Diazotrophic assemblages from the majority of the water column samples from the Mid- and South Bay clustered with the sediment assemblage in Mid-Bay. Dissolved inorganic nitrogen, salinity, dissolved organic carbon and dissolved organic phosphorus had a significant relationship with the diazotrophic bacterioplankton community. Higher diversity in the freshwater end of the system may reflect variability in disturbance rates and environmental conditions such as forms and concentrations of organic matter, nutrients and oxygen.     

Identification of repertoires of surface antigens on leukemias using an antibody microarray

We have previously described a microarray of cluster of differentiation (CD) antibodies that enables concurrent determination of more than 60 CD antigens on leukocytes. This procedure does not require protein purification or labeling, or a secondary detection system. Whole cells are captured by a microarray of 10 nL antibody dots immobilized on a nitrocellulose film on a microscope slide. Distinct patterns of cell binding are observed for different leukemias or lymphomas. These haematological malignancies arise from precursor cells of T- or B-lymphocytic, or myeloid lineages of hematopoiesis. The dot patterns obtained from patients are distinct from those of peripheral blood leukocytes from normal subjects. This microarray technology has recently undergone a number of refinements. The microarray now contains more CD antibodies, and a scanner for imaging dot patterns and software for data analysis provide an extensive immunophenotype sufficient for diagnosis of common leukemias. The technology is being evaluated for diagnosis of leukemias with parallel use of conventional diagnostic criteria.     

Cigarette smoke-promoted acquisition of bacterial pathogens in the upper respiratory tract leads to enhanced inflammation in mice

Background

Bacterial colonization and recurrent infections of the respiratory tract contribute to the progression of chronic obstructive pulmonary disease (COPD). There is evidence that exacerbations of COPD are provoked by new bacterial strains acquired from the environment. Using a murine model of colonization, we examined whether chronic exposure to cigarette smoke (CS) promotes nasopharyngeal colonization with typical lung pathogens and whether colonization is linked to inflammation in the respiratory tract.

Methods

C57BL/6 N mice were chronically exposed to CS. The upper airways of mice were colonized with nontypeable Haemophilus influenzae (NTHi) or Streptococcus pneumoniae. Bacterial colonization was determined in the upper respiratory tract and lung tissue. Inflammatory cells and cytokines were determined in lavage fluids. RT-PCR was performed for inflammatory mediators.

Results

Chronic CS exposure resulted in significantly increased numbers of viable NTHi in the upper airways, whereas NTHi only marginally colonized air-exposed mice. Colonization with S. pneumoniae was enhanced in the upper respiratory tract of CS-exposed mice and was accompanied by increased translocation of S. pneumoniae into the lung. Bacterial colonization levels were associated with increased concentrations of inflammatory mediators and the number of immune cells in lavage fluids of the upper respiratory tract and the lung. Phagocytosis activity was reduced in whole blood granulocytes and monocytes of CS-exposed mice.

Conclusions

These findings demonstrate that exposure to CS impacts the ability of the host to control bacterial colonization of the upper airways, resulting in enhanced inflammation and susceptibility of the host to pathogens migrating into the lung.     

Development of an oligonucleotide microarray method for Salmonella serotyping

Adequate identification of Salmonella enterica serovars is a prerequisite for any epidemiological investigation. This is traditionally obtained via a combination of biochemical and serological typing. However, primary strain isolation and traditional serotyping is time‐consuming and faster methods would be desirable. A microarray, based on two housekeeping and two virulence marker genes (atpD, gyrB, fliC and fljB), has been developed for the detection and identification of the two species of Salmonella (S. enterica and S. bongori), the five subspecies of S. enterica (II, IIIa, IIIb, IV, VI) and 43 S. enterica ssp. enterica serovars (covering the most prevalent ones in Austria and the UK). A comprehensive set of probes (n = 240), forming 119 probe units, was developed based on the corresponding sequences of 148 Salmonella strains, successfully validated with 57 Salmonella strains and subsequently evaluated with 35 blind samples including isolated serotypes and mixtures of different serotypes. Results demonstrated a strong discriminatory ability of the microarray among Salmonella serovars. Threshold for detection was 1 colony forming unit per 25 g of food sample following overnight (14 h) enrichment.     

High-throughput identification of genetic markers using representational oligonucleotide microarray analysis

We describe a novel approach for high-throughput development of genetic markers using representational oligonucleotide microarray analysis. We test the performance of the method in sugar beet (Beta vulgaris L.) as a model for crop plants with little sequence information available. Genomic representations of both parents of a mapping population were hybridized on microarrays containing in total 146,554 custom made oligonucleotides based on sugar beet bacterial artificial chromosome (BAC) end sequences and expressed sequence tags (ESTs). Oligonucleotides showing a signal with one parental line only, were selected as potential marker candidates and placed onto an array, designed for genotyping of 184 F₂ individuals from the mapping population. Utilizing known co-dominant anchor markers we obtained 511 new dominant markers (392 derived from BAC end sequences, and 119 from ESTs) distributed over all nine sugar beet linkage groups and calculated genetic maps. Further improvements for large-scale application of the approach are discussed and its feasibility for the cost-effective and flexible generation of genetic markers is presented.     

Ferrocene conjugated oligonucleotide for electrochemical detection of DNA base mismatch

We describe the synthesis, binding, and electrochemical properties of ferrocene-conjugated oligonucleotides (Fc-oligos). The key step for the preparation of Fc-oligos contains the coupling of vinylferrocene to 5-iododeoxyuridine via Heck reaction. The Fc-conjugated deoxyuridine phosphoramidite was used in the Fc-oligonucleotide synthesis. We show that thiol-modified Fc-oligos deposited onto gold electrodes possess potential ability in electrochemical detection of DNA base mismatch.