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


RESULTS
Comparison 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.


CONCLUSIONS
The assay rapidly provides reliable data, which can guide optimal antimicrobial treatment decisions in a timely manner.
Järvinen
Anna-Kaarina
AK
Mobidiag Ltd, 00290 Helsinki, Finland. anna-kaarina.jarvinen@mobidiag.com
Laakso
Sanna
S
Piiparinen
Pasi
P
Aittakorpi
Anne
A
Lindfors
Merja
M
Huopaniemi
Laura
L
Piiparinen
Heli
H
Mäki
Minna
M
D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics*; D027081:DNA Gyrase / Q000235:genetics; D017931:DNA Primers; D027101:DNA Topoisomerase IV / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase