GreeneChip Design and Fabrication

Viral probes were designed to represent a minimum of 3 distinct genomic target regions for every family or genus of vertebrate virus in the ICTVdB. When possible, we chose highly conserved regions within a coding sequence for an enzyme such as a polymerase and 2 other regions that corresponded to more variable structural proteins. We thought that RNAs that encode structural proteins may be present at higher levels than those that encode proteins needed only in catalytic amounts and that use of probes representing noncontiguous sites along the genome might allow detection of naturally occurring or intentionally created chimeric viruses.

Any diagnostic tool based on nucleic acid hybridization is necessarily dependent on the extent to which probes are complementary to their targets. Although sequence databases are increasingly comprehensive, it is unlikely that more than a fraction of the existing microbial sequence space has been explored. Our intent in implementing GreeneChip was to have the potential to identify known and related agents for which precise sequence information was not available. To assess the extent to which a given probe sequence can hybridize to a nonmatching but related sequence, we analyzed synthetic mismatch controls. Whereas up to 15 terminal mismatches had little effect, strings of >5 mismatches distributed throughout a sequence, particularly mismatched G/C pairs, resulted in reduced signal; >12 mismatches distributed throughout a sequence resulted in no signal. On the basis of these findings, we pursued a conservative strategy in array design wherein a viral sequence was considered to be covered only if the array included at least 1 complementary probe with <5 mismatches.

The process for identifying bacterial, fungal, and parasitic probes was similar, although restricted to 16S and 18S rRNA sequences. Viral (GreeneChipVr) and panmicrobial (GreeneChipPm) array platforms were based on the GreeneVrdB and GreenePmdB, respectively. GreeneChipVr version 1.0 contained 9,477 probes to address all vertebrate viruses in the integrated ICTV/NCBI database (1,710 species, including all reported isolates) in 3 gene regions with <5 nucleotide mismatches. GreeneChipPm version 1.0 contained 29,495 probes that included probes comprising GreeneChipVr version 1.0, as well as 11,479 16S rRNA bacterial probes, 1,120 18S rRNA fungal probes, and 848 18S rRNA parasite probes. A total of 300 host immune response probes were added to arrays as a potential index to pathogenesis.

The 60-mer oligonucleotide arrays were synthesized on 70-mm × 20-mm glass slides by using an inkjet deposition system (Agilent Technologies, Palo Alto, CA, USA). A slide can accept up to 244,000 different 60-mer probes or 8 arrays, each comprising >15,000 probes. To facilitate alignment during scanning, 1,000 additional landing-light probes (5′-ATC ATC GTA GCT GGT CAG TGT ATC CTT TTT TTT TTA TCA TCG TAG CTG GTC AGT GTA TCC-3′) were placed in the corners and in a grid on the array. Fluorescently labeled synthetic oligonucleotides complementary to the control probes were included in all hybridizations.

Viruses and Clinical Samples

Sources of viruses and viral reference strains used in this study are shown in Tables 1 and ​and2.2. Blood sample 200501379 (Lake Victoria marburgvirus, reference sample from Angola, 2005) and blood sample Angola-460 from a patient suspected of having viral hemorrhagic fever (VHF) were received in containers approved by the International Air Transport Association at either the Centers for Disease Control and Prevention in Atlanta, Georgia, USA or the Public Health Agency of Canada in Winnipeg, Ontario, Canada, respectively.

Sources of clinical samples are shown in Table 3. Nasopharyngeal aspirates (SO4606 and SO5265) were collected by the Instituto de Salud Carlos III in Madrid, Spain, from children with respiratory disease. We also analyzed a nasopharyngeal aspirate (sample 23), a postmortem specimen from a patient who died of infection with severe acute respiratory syndrome coronavirus (SARS-CoV, sample TM-167), urine specimens from 2 patients with urinary tract infections (samples CUMC-NR7 and CUMC-NR9), a urine specimen from an asymptomatic patient (sample CUMC-LO1), and endometrial and lung tissues from a patient infected with Mycobacterium tuberculosis (samples CUMC-DL1 and CUMC-DL3).

Sample Preparation and GreeneChip Hybridization

RNA was isolated from blood of VHF patients by using a 6100 Nucleic Acid PrepStation (Applied Biosystems, Foster City, CA, USA). RNA from virus isolates (culture supernatant) and other clinical samples (blood, nasopharyngeal aspirate, tissue, urine) was isolated by using the Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH, USA). DNA was removed from RNA preparations by treatment with DNase I (DNA-free, Ambion Inc., Austin, TX, USA). First-strand reverse transcription was initiated with a random octamer linked to a specific primer sequence (5′-GTT TCC CAG TAG GTC TCN NNN NNN N-3′) (5). After digestion with RNase, cDNA was amplified by using a 1:9 mixture of the above primer and a primer targeting the specific primer sequence (5′-CGC CGT TTC CCA GTA GGT CTC-3′). Initial PCR amplification cycles were performed at a low annealing temperature (25°C); subsequent cycles used a stringent annealing temperature (55°C) to favor priming through the specific sequence. Products of this first PCR were then labeled in a subsequent PCR with the specific primer sequence linked to a capture sequence for 3 DNA dendrimers containing >300 fluorescent reporter molecules (Genisphere Inc., Hatfield, PA, USA), Products of the second PCR were added to sodium dodecyl sulfate–based hybridization buffer (Genisphere Inc.), heated for 10 min at 80°C, and added to GreeneChip for hybridization for 16 h at 65°C. After 10-min washes at room temperature with 6 × SSC (0.9 mol/L NaCl, 0.09 mol/L sodium citrate, pH 7.0), 0.005% Triton X-100, and 0.1 × SSC, 0.005% Triton X-100, Cy3 3DNA dendrimers were added and incubated at 65°C for 1 h. Slides were washed as before, air dried, and scanned (DNA Microarray scanner, Agilent Technologies).

GreeneChip Analysis

Log-transformed analysis of microarrays using p values (GreeneLAMP) version 1.0 software was created to assess results of GreeneChip hybridizations. A map built from BLAST data was used to connect probe sequences to the respective entries in the GreenePmdB. Each of those sequences corresponds to an NCBI Taxonomy ID (TaxID). Individual TaxIDs were mapped to nodes in a taxonomic tree built based on ICTV virus taxonomy or the NCBI taxonomic classification for other organisms. The program output is a ranked list of candidate TaxIDs.

Probe intensities were corrected for background, log₂-transformed, and converted to Z scores (and their corresponding p values). Where available, control-matched experiments from uninfected samples were used, and spots >2 standard deviations from the mean were subtracted. In instances where control-matched samples were not available, the background distribution of signal fluorescence in an array was calculated by using fluorescence associated with 1,000 random 60 mers (null probes). In both scenarios, positive events were selected by applying a false-positive rate of 0.01 (the rate at which null probes are scored as significant) and a minimum p value per probe of 0.1 in cases with a matching control and 0.023 (2 standard deviations) in cases without a matching control. Candidate TaxIDs were ranked by combining the p values for the positive probes for that TaxID by using the QFAST method of Bailey and Gribskov (9). This approach makes the following assumptions: 1) spot intensities are normally distributed; 2) spots represent independent observations (to minimize this effect clustering is used to collapse probes that are 95% identical); and 3) there are relatively few (<100) positive probes for any given TaxID. Probes for each kingdom (bacteria, eukaryotes, fungi, viruses) were analyzed independently to compensate for variations in signal-to-noise levels.

Sequence Recovery from Hybridized Arrays

When a hybridization signal suggests a novel or chimeric agent, or the investigator wants to obtain sequence information, cDNA can be eluted for amplification and sequence analysis. A total of 100 μL of water at 90°C is added to the array and pipetted up and down 10 times. The eluate is recovered, amplified with the specific primer used during the initial amplification, and cloned into a plasmid vector (TOPO TA, Invitrogen, Carlsbad, CA, USA). After transformation into Escherichia coli, colonies are screened by sequencing. Primers based on the obtained sequence can be designed for confirmation of the agent or for specific (real-time) PCR screening of other specimens.

We thank Mady Hornig for helpful comments and providing host immune response probes and David Smith, David Boyle, Phyllis Della-Latta, Adolfo Garcia-Sastre, Gerry Harnett, Phillipa Jack, Cheryl Johansen, Anthony Mazzuli, John Mackenzie, Hendrik Nollens, Pilar Perez-Breña, and David Williams for specimens used in assay development and validation. We dedicate this paper to Allan Rosenfield, a humanitarian and visionary in global health.

The study was supported by National Institutes of Health grants AI51292, AI056118, AI55466, U54AI57158 (Northeast Biodefense Center-Lipkin), and U01AI070411, and the Ellison Medical Foundation.