Panmicrobial oligonucleotide array for diagnosis of infectious diseases

Abstract

To facilitate rapid, unbiased, differential diagnosis of infectious diseases, we designed GreeneChipPm, a panmicrobial microarray comprising 29,455 sixty-mer oligonucleotide probes for vertebrate viruses, bacteria, fungi, and parasites. Methods for nucleic acid preparation, random primed PCR amplification, and labeling were optimized to allow the sensitivity required for application with nucleic acid extracted from clinical materials and cultured isolates. Analysis of nasopharyngeal aspirates, blood, urine, and tissue from persons with various infectious diseases confirmed the presence of viruses and bacteria identified by other methods, and implicated Plasmodium falciparum in an unexplained fatal case of hemorrhagic feverlike disease during the Marburg hemorrhagic fever outbreak in Angola in 2004-2005.

Figure 1

A) Signal intensity for viral probes in blood sample 200501379. Probe intensities were background corrected, log₂-transformed, and converted to Z scores (and corresponding p values). Background distribution of signal fluorescence in the array was calculated by using fluorescence associated with 1,000 random null probes. Positive events were selected by applying a minimum p value per probe of 0.023 (2 standard deviations). Analysis of positive events with GreeneLAMP resulted in prediction of TaxID sample 11269 as the top prediction by the combined p value. Solid squares indicate Lake Victoria marburgvirus probes; open triangles indicate other probes. Ten of (90.9%) of 11 Lake Victoria marburgvirus probes were positive. B) Genomic location of positive Lake Victoria marburgvirus probes. Sequence-based analysis identified GenBank accession no. DQ447653 (Lake Victoria marburgvirus–Angola2005 strain Ang1379c) with 10 positive probes (all 8 motifs) as the best match.

Figure 2

Analysis of 18S rRNA sequence (nt 291,256–292,364) recovered from the array after hybridization of sample Angola-460. The phylogenetic tree was reconstructed with the neighbor-joining method applying a Kimura 2-parameter model with MEGA version 3.1 (16). Number of nucleotide substitutions per site are indicated by the scale bar; bootstrap values (percentage of 1,000 pseudoreplicates) are given at relevant branches. Circles indicate Plasmodium falciparum sequences; inverted triangles indicate other known plasmodial pathogens of humans.