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

ABSTRACT: 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. CONCLUSION: The assay rapidly provides reliable data, which can guide optimal antimicrobial treatment decisions in a timely manner.

SUBMITTER: Jarvinen AK

PROVIDER: S-EPMC2741468 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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

Järvinen Anna-Kaarina AK Laakso Sanna S Piiparinen Pasi P Aittakorpi Anne A Lindfors Merja M Huopaniemi Laura L Piiparinen Heli H Mäki Minna M

BMC microbiology 20090810

<h4>Background</h4>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. ...[more]

PMID: 19664269

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Project description:Respiratory and enteric diseases continue to be two major causes of economic losses to the cattle industry worldwide. Despite their multifactorial etiology, the currently available diagnostic tests for bovine respiratory disease complex (BRDC) and bovine enteric disease (BED) are single-pathogen-tests. DNA microarray when combined with multiplex polymerase chain reaction (PCR) is a powerful tool in detection and differentiation of multiple pathogens in a single sample. This study reports development and initial validation of two independent highly sensitive and specific multiplex PCR-electronic microarray assays, one for the detection and differentiation of pathogens of the BRDC and the other for detection and differentiation of pathogens of the BED. The BRDC multiplex PCR-microarray assay was able to detect and differentiate four bacteria (Mannheimia haemolytica, Histophilus somni, Pasteurella multocida, and Mycoplasma bovis) and five viruses [bovine parainfluenza virus-3, bovine respiratory syncytial virus, bovine herpesvirus-1, bovine coronavirus (BCoV), and bovine viral diarrhea virus (BVDV)] associated with BRDC. The BED multiplex PCR- microarray- assay was able to detect and differentiate four bacteria (Clostridium perfringens, Escherichia coli, Salmonella enterica Dublin, and Salmonella enterica Typhimurium), three protozoa (Eimeria zuernii, Eimeria bovis, and Cryptosporidium parvum), and four viruses (bovine torovirus, bovine rotavirus, BCoV, and BVDV) associated with the BED. Both assays detected their respective targets individually or in combination when present. The limit-of-detection of each assay at the PCR amplification and DNA microarray levels was determined using previously titrated laboratory amplified target pathogens or using quantified synthetic nucleotides. Both assays showed very high analytical sensitivity and specificity, and were validated using a limited number of clinical samples. The BRDC and BED multiplex PCR- microarray-assays developed in this study, with further clinical validation, could be used in veterinary diagnostic laboratories for the rapid and simultaneous identification of pathogens to facilitate quick and accurate decision making for the control and treatment of these two economically important disease complexes. Furthermore, these assays could be very effective tools in epidemiological studies as well as for screening of healthy animals to identify carriers that may potentially develop BRDC or BED.

Dataset Information

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

Publications

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

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