[Show abstract][Hide abstract] ABSTRACT: A resequencing microarray called PathogenID v2.0 has been developed and used to explore various strategies of sequence selection for its design. The part dedicated to influenza viruses was based on consensus sequences specific for one gene generated from global alignments of a large number of influenza virus sequences available in databanks.
For each HA (H1, H2, H3, H5, H7 and H9) and NA (N1, N2 and N7) molecular type chosen to be tested, 1 to 3 consensus sequences were computed and tiled on the microarray. A total of 12 influenza virus samples from different host origins (humans, pigs, horses and birds) and isolated over a period of about 50 years were used in this study. Influenza viruses were correctly identified, and in most cases with the accurate information of the time of their emergence.
PathogenID v2.0 microarray demonstrated its ability to type and subtype influenza viruses, often to the level of viral variants, with a minimum number of tiled sequences. This validated the strategy of using consensus sequences, which do not exist in nature, for our microarray design. The versatility, rapidity and high discriminatory power of the PathogenID v2.0 microarray could prove critical to detect and identify viral genome reassortment events resulting in a novel virus with epidemic or pandemic potential and therefore assist health authorities to make efficient decisions about patient treatment and outbreak management.
[Show abstract][Hide abstract] ABSTRACT: The rapid and accurate identification of pathogens is critical in the control of infectious disease. To this end, we analyzed the capacity for viral detection and identification of a newly described high-density resequencing microarray (RMA), termed PathogenID, which was designed for multiple pathogen detection using database similarity searching. We focused on one of the largest and most diverse viral families described to date, the family Rhabdoviridae. We demonstrate that this approach has the potential to identify both known and related viruses for which precise sequence information is unavailable. In particular, we demonstrate that a strategy based on consensus sequence determination for analysis of RMA output data enabled successful detection of viruses exhibiting up to 26% nucleotide divergence with the closest sequence tiled on the array. Using clinical specimens obtained from rabid patients and animals, this method also shows a high species level concordance with standard reference assays, indicating that it is amenable for the development of diagnostic assays. Finally, 12 animal rhabdoviruses which were currently unclassified, unassigned, or assigned as tentative species within the family Rhabdoviridae were successfully detected. These new data allowed an unprecedented phylogenetic analysis of 106 rhabdoviruses and further suggest that the principles and methodology developed here may be used for the broad-spectrum surveillance and the broader-scale investigation of biodiversity in the viral world.
Journal of Virology 09/2010; 84(18):9557-74. DOI:10.1128/JVI.00771-10 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phi29 polymerase based amplification methods provides amplified DNA with minimal changes in sequence and relative abundance for many biomedical applications. RNA virus detection using microarrays, however, can present a challenge because phi29 DNA polymerase cannot amplify RNA nor small cDNA fragments (<2000 bases) obtained by reverse transcription of certain viral RNA genomes. Therefore, ligation of cDNA fragments is necessary prior phi29 polymerase based amplification. We adapted the QuantiTect Whole Transcriptome Kit (Qiagen) to our purposes and designated the method as Whole Transcriptome Amplification (WTA).
WTA successfully amplified cDNA from a panel of RNA viruses representing the diversity of ribovirus genome sizes. We amplified a range of genome copy numbers from 15 to 4 x 10(7) using WTA, which yielded quantities of amplified DNA as high as 1.2 microg/microl or 10(10) target copies. The amplification factor varied between 10(9) and 10(6). We also demonstrated that co-amplification occurred when viral RNA was mixed with bacterial DNA.
This is the first report in the scientific literature showing that a modified WGA (WTA) approach can be successfully applied to viral genomic RNA of all sizes. Amplifying viral RNA by WTA provides considerably better sensitivity and accuracy of detection compared to random RT-PCR.
[Show abstract][Hide abstract] ABSTRACT: Identification of microbial pathogens in clinical specimens is still performed by phenotypic methods that are often slow and cumbersome, despite the availability of more comprehensive genotyping technologies. We present an approach based on whole-genome amplification and resequencing microarrays for unbiased pathogen detection. This 10 h process identifies a broad spectrum of bacterial and viral species and predicts antibiotic resistance and pathogenicity and virulence profiles. We successfully identify a variety of bacteria and viruses, both in isolation and in complex mixtures, and the high specificity of the microarray distinguishes between different pathogens that cause diseases with overlapping symptoms. The resequencing approach also allows identification of organisms whose sequences are not tiled on the array, greatly expanding the repertoire of identifiable organisms and their variants. We identify organisms by hybridization of their DNA in as little as 1-4 h. Using this method, we identified Monkeypox virus and drug-resistant Staphylococcus aureus in a skin lesion taken from a child suspected of an orthopoxvirus infection, despite poor transport conditions of the sample, and a vast excess of human DNA. Our results suggest this technology could be applied in a clinical setting to test for numerous pathogens in a rapid, sensitive and unbiased manner.
[Show abstract][Hide abstract] ABSTRACT: Unbiased and simultaneous identification of viral and bacterial pathogens in clinical samples can influence healthcare management decisions and improve medical care. Here we report the utility of microarray technology for rapid identification and characterization of pathogen mixtures in clinical samples. A DNA resequencing microarray was designed for the identification of 28 bacterial pathogens and 42 viruses on the NIAID priority list, 264 antibiotic resistance genes (ABR) and 256 toxins and others. This array contains 2.56 million unique probes which allow resequencing of 300,000 bp in a single experiment. Organism identification can be achieved by resequencing several conserved genes, including rrs (16S rRNA), rpoB, gyrA, gyrB, viral polymerases. A clinical sample from a human skin swab was tested using the resequencing array, and sequence analysis of conserved genes indicated the presence of Monkeypox virus, human mitochondrial DNA, Staphylococcus aureus and genes conferring MLS and tetracycline resistance. These results indicate that microarray-based assays can be used to accurately detect mixtures of viral, bacterial, and host DNA. To determine the effect of contaminating human material, we tested S. aureus containing the parC, aac6-aph2, and tetM ABR genes, in combination with 10μg human genomic DNA. Results show that S. aureus and ABR genes can be identified accurately after 1 hour of hybridization to the microarray even in the presence of an excess of human DNA. Data indicate that the array can accurately identify closely related species, even if the exact organism sequence is not tiled on the array. This approach is promising for rapid and unbiased characterization of known and unknown pathogens in clinical specimens.
Infectious Diseases Society of America 2006 Annual Meeting; 10/2006