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Rapid Identification of Bacterial Pathogens using a PCR- and Microarray-Based Assay

ABSTRACT: A method based on a modified broad-range PCR and an oligonucleotide microarray for the simultaneous detection and identification of 12 bacterial pathogens at the species level.

ORGANISM(S): Acinetobacter baumannii Streptococcus pneumoniae Klebsiella pneumoniae Neisseria meningitidis Enterococcus faecalis Homo sapiens unidentified Streptococcus agalactiae Staphylococcus epidermidis Listeria monocytogenes Staphylococcus aureus Streptococcus pyogenes Haemophilus influenzae Enterococcus faecium

PROVIDER: GSE17221 | GEO | 2009/08/31

SECONDARY ACCESSION(S): PRJNA119185

REPOSITORIES: GEO

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Rapid Identification of Bacterial Pathogens using a PCR- and Microarray-Based Assay

Project description:A method based on a modified broad-range PCR and an oligonucleotide microarray for the simultaneous detection and identification of 12 bacterial pathogens at the species level. The feasibility of this assay in routine diagnostic testing was evaluated using 146 blood culture positive and 40 blood culture negative samples. The processed data are composed of ORGANISM NAME or NEGATIVE answer depending if the pathogen is detected in the sample or not. See supplementary file linked below.

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Rapid Identification of Bacterial Pathogens using a PCR- and Microarray-Based Assay

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Rapid identification of bacterial pathogens using a PCR- and microarray-based assay.

Project description:BackgroundDuring the course of a bacterial infection, the rapid identification of the causative agent(s) is necessary for the determination of effective treatment options. We have developed a method based on a modified broad-range PCR and an oligonucleotide microarray for the simultaneous detection and identification of 12 bacterial pathogens at the species level. The broad-range PCR primer mixture was designed using conserved regions of the bacterial topoisomerase genes gyrB and parE. The primer design allowed the use of a novel DNA amplification method, which produced labeled, single-stranded DNA suitable for microarray hybridization. The probes on the microarray were designed from the alignments of species- or genus-specific variable regions of the gyrB and parE genes flanked by the primers. We included mecA-specific primers and probes in the same assay to indicate the presence of methicillin resistance in the bacterial species. The feasibility of this assay in routine diagnostic testing was evaluated using 146 blood culture positive and 40 blood culture negative samples.ResultsComparison of our results with those of a conventional culture-based method revealed a sensitivity of 96% (initial sensitivity of 82%) and specificity of 98%. Furthermore, only one cross-reaction was observed upon investigating 102 culture isolates from 70 untargeted bacteria. The total assay time was only three hours, including the time required for the DNA extraction, PCR and microarray steps in sequence.ConclusionThe assay rapidly provides reliable data, which can guide optimal antimicrobial treatment decisions in a timely manner.

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