Rapid pathogen identification using a novel microarray-based assay with purulent meningitis in cerebrospinal fluid

Abstract

In order to improve the diagnosis of pathogenic bacteria in cerebrospinal fluid (CSF) with purulent meningitis, we developed a DNA microarray technique for simultaneous detection and identification of seven target bacterium. DNA were extracted from 24 CSF samples with purulent meningitis (or suspected purulent meningitis). The specific genes of each pathogen were chosen as the amplification target, performed the polymerase chain reaction (PCR), labeled with a fluorescence dye, and hybridized to the oligonucleotide probes on the microarray. There is no significant cross-hybridization fluorescent signal occurred in untargeted bacteria. There were 87.5% (21/24) positive results in DNA microarray compared with the 58.3% (14/24) of the CSF culture test. Of which 58.3% (14/24) of the patients with culture-confirmed purulent meningitis, 37.5% (9/24) patients who were not confirmed by culture test but were demonstrated by the clinical diagnosis and DNA microarray. Multiple bacterial infections were detected in 5 cases by the microarray. In addition, the number of gene copies was carried out to determine the sensitivity of this technique, which was shown to be 3.5 × 10¹ copies/μL. The results revealed that the microarray technique which target pathogens of the CSF specimen is better specificity, accuracy, and sensitivity than traditional culture method. The microarray method is an effective tool for rapidly detecting more target pathogens and identifying the subtypes of strains which can eliminate the impact of the different individuals with purulent meningitis for prompt diagnosis and treatment.

Figure 1

Oligonucleotide probes for array positioning.

Figure 2

The specificity and sensitivity of the pathogen probes. (A) Microarray hybridized with the probe untarget bacteria and human genome DNA. (B) Microarray hybridized with the probe target bacteria. NC means microarray hybridized with ddH₂O and positive control sequence (show the signal of PC probes). (C) Microarray hybridized with the Escherichia coli (ATCC 25922) which diluted for concentration gradient (the original DNA samples were extracted from CSF).

Figure 3

The gel images of PCR products using specific primers. (A) PCR products with 6 standard strains using specific primers. (B) PCR products with 2 specimens which was chosen randomly from all specimens.

Figure 4

The results of microarray hybridization of 24 specimens from CSF. (A) There is a probe repeat for every 3 spots and “or” relation between all probes for the same bacteria (multiple probes were used for detecting bacteria subtypes). So the signals of three spots (or its multiples) means the sample containing this target bacteria.