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

Lee, Dae-Young; Shannon, Kelly; Beaudette, Lee A.

doi:10.1016/j.mimet.2005.09.008

Cited by 142 publications

(93 citation statements)

References 39 publications

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“…The arrays have the ability to perform a series of simultaneous hybridization assays that can be easily interpreted. Hybridization patterns of DNA sequences with specific probes indicate the presence or absence of specific microorganisms in the sample (12)(13)(14). DNA microarrays are developed to detect and identify a large number of bacteria present in food, blood, stool and urine (15)(16)(17).…”

Section: Introductionmentioning

confidence: 99%

Simultaneous Detection of Escherichia coli, Salmonella enterica, Listeria monocytogenes and Bacillus cereus by Oligonucleotide Microarray

Sarshar¹,

Shahrokhi²,

Ranjbar³

et al. 2015

Int J Enteric Pathog

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Background:Traditional laboratory methods to detect pathogenic bacteria are time consuming and laborious. Therefore, it is essential to use powerful and reliable molecular methods for quick and simultaneous detection of microbial pathogens. Objectives: The current study aimed to evaluate the capability and efficiency of 23S rDNA sequence for rapid and simultaneous detection of four important food-borne pathogens by an oligonucleotide microarray technique. Materials and Methods:The 23S rDNA sequences of Escherichia coli, Salmonella enterica, Listeria monocytogenes and Bacillus cereus were obtained from GenBank databases and used to design the oligonucleotide probes and primers by Vector NTI software. Oligonucleotide probes were placed on a nylon membrane and hybridization was performed between probes and 23S rDNA digoxigenin-labeled polymerase chain reaction (PCR) products. Hybridization signals were visualized by NBT/BCIP color development. Results: Positive hybridization color was produced for Escherichia coli, Salmonella enterica, Listeria monocytogenes and Bacillus cereus. The oligonucleotide microarray detected all bacterial strains in a single reaction in less than five hours. The sensitivity of the performed microarray assay was 10 3 cfu/mL for each species of pathogen. No cross reaction was found between the tested bacterial species. Conclusions: The obtained results indicated that amplification of 23S rDNA gene followed by oligonucleotide microarray hybridization is a rapid and reliable method to identify and discriminate foodborne pathogens tested under the study.

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Section: Introductionmentioning

confidence: 99%

Simultaneous Detection of Escherichia coli, Salmonella enterica, Listeria monocytogenes and Bacillus cereus by Oligonucleotide Microarray

Sarshar¹,

Shahrokhi²,

Ranjbar³

et al. 2015

Int J Enteric Pathog

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“…5 For microorganism detection, DNA microarrays can also be used for the quantification in food or water samples. 6,7 For these analytical purposes, fluorescence, electrochemical, chemiluminescence (CL) and label-free microarray read-out systems have been developed. [8][9][10][11][12][13][14] CL microarrays use the effect of light emission caused by a chemical reaction with the assistance of an enzyme label.…”

Section: Introductionmentioning

confidence: 99%

Quantification of E. coli DNA on a Flow-through Chemiluminescence Microarray Readout System after PCR Amplification

Donhauser

Seidel

2009

ANAL. SCI.

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We report on a hybridization assay using DNA microarrays for the quantification of amplification products of the uidA gene of E. coli. Using the stopped-PCR strategy, the amplified target DNA was strongly dependent on the applied gene copies. The quantification was carried out by a flow-through chemiluminescence microarray readout system. The DNA microarrays were based on a poly(ethylene glycol)-modified glass substrate. The probes on the surface were 18 or 25 nucleotides long and the quantified PCR product was 60 nucleotides. The amplification was stopped after 25 cycles; at this point amplification was in the middle of the logarithmical phase, and the spread between different DNA starting concentrations reached the maximum. A conjugate of streptavidin and horseradish peroxidase (HRP) bound to the biotinylated strands on the microarray surface and catalyzed the reaction of luminol and hydrogen peroxide. The generated light emission was recorded by a sensitive charge-coupled device (CCD) camera. The detection limit for the gene uidA (b-galactosidase) of E. coli was 1.1 ¥ 10 5 copies/mL. This system allowed for a sensitive detection and quantification of E. coli in a concentration range from 10 6 to 10 9 copies/mL.

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“…Microarray technology has been applied successfully to study microbial commu-nities in several habitats such as municipal wastewater [29], landfill methanotroph communities [43], and trinitrotoluenecontaminated soils [17]. Clone library studies and metagenome approaches like pyrosequencing yield information beyond the cultivated part of the community; however, these techniques involve much work and are expensive.…”

Section: Introductionmentioning

confidence: 99%

Application of COMPOCHIP Microarray to Investigate the Bacterial Communities of Different Composts

et al. 2008

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A microarray spotted with 369 different 16S rRNA gene probes specific to microorganisms involved in the degradation process of organic waste during composting was developed. The microarray was tested with pure cultures, and of the 30,258 individual probe-target hybridization reactions performed, there were only 188 false positive (0.62%) and 22 false negative signals (0.07%). Labeled target DNA was prepared by polymerase chain reaction amplification of 16S rRNA genes using a Cy5-labeled universal bacterial forward primer and a universal reverse primer. The COMPOCHIP microarray was applied to three different compost types (green compost, manure mix compost, and anaerobic digestate compost) of different maturity (2, 8, and 16 weeks), and differences in the microorganisms in the three compost types and maturity stages were observed. Multivariate analysis showed that the bacterial composition of the three composts was different at the beginning of the composting process and became more similar upon maturation. Certain probes (targeting Sphingobacterium, Actinomyces, Xylella/Xanthomonas/ Stenotrophomonas, Microbacterium, Verrucomicrobia, Planctomycetes, Low G + C and Alphaproteobacteria) were more influential in discriminating between different composts. Results from denaturing gradient gel electrophoresis supported those of microarray analysis. This study showed that the COMPOCHIP array is a suitable tool to study bacterial communities in composts.

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

Cited by 142 publications

References 39 publications

Simultaneous Detection of Escherichia coli, Salmonella enterica, Listeria monocytogenes and Bacillus cereus by Oligonucleotide Microarray

Simultaneous Detection of Escherichia coli, Salmonella enterica, Listeria monocytogenes and Bacillus cereus by Oligonucleotide Microarray

Quantification of E. coli DNA on a Flow-through Chemiluminescence Microarray Readout System after PCR Amplification

Application of COMPOCHIP Microarray to Investigate the Bacterial Communities of Different Composts

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