In a recent publication [1], Boachuan Lin and Zheng Wang in Dr. David Stenger's laboratory (Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, D.C.) reported the feasibility of using microarray technology for surveillance and diagnosis of respiratory pathogens from clinical samples. Ambion’s MICROBEnrich technology contributed to the success of the study by depleting nucleic acids isolated from clinical samples of mammalian sequences, thereby enriching the samples for the pathogen-derived RNA they sought to detect. Here is a brief overview of this publication.

Widespread concern about both bioterrorism and natural pathogens was the impetus for these researchers to address the need for advanced diagnosis and surveillance techniques for infectious diseases. Their approach sought to take advantage of the rapidly increasing amount of available pathogen sequence information and the power of microarray hybridization technology that can query for 105 to 106 sequences simultaneously. By optimizing sample amplification, probe specificity, and the data interpretation strategy, the researchers demonstrate the feasibility of using a microarray approach for broad spectrum surveillance of respiratory pathogens at clinically relevant sensitivity levels.

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Nucleic Acid Purification and Amplification

A key strategy employed by the group was optimization of DNA and RNA enrichment and amplification procedures to eliminate the necessity of using pathogen-specific amplification scenarios that had been employed in previous studies [2, 3]. To prepare nucleic acid samples for amplification, pathogen-derived DNA was enriched using a Cot Human DNA subtraction technique, and RNA was enriched using Ambion’s MICROBEnrich Kit. The pathogen-enriched nucleic acid was amplified using a whole-genome [4] or random amplification method [5, 6].

Microarray Probe Design and Data Interpretation

The authors designed a resequencing array consisting of a set of tiled probes to regions of pathogen RNA or DNA selected for both efficient hybridization and unique identification of pathogenic subspecies. Pathogens of more urgent concern were represented on the arrays in even more detail to permit detection of clinically relevant strains. The use of these microarrays, called RPM v.1, combined with unambiguous statistical methods for data analysis, enabled simultaneous detection of respiratory pathogens without requiring pathogen-specific amplification or comparison to fixed hybridization patterns (a strategy that is ineffective because of the preponderance of sequence anomalies in viral sequences from clinical samples).

References

  1. Lin B, Wang Z, Vora GJ, Thornton JA, Schnur JM, Thach DC, Blaney KM, Ligler AG, Malanoski AP, Santiago J, Walter EA, Agan BK, Metzgar D, Seto D, Daum LT, Kruzelock R, Rowley RK, Hanson EH, Tibbetts C, Stenger DA (2006) Broad-spectrum respiratory tract pathogen identification using resequencing DNA microarrays. Genome Res 16(4):527–35.

  2. López MM, Bertolini E, Olmos A, Caruso P, Gorris MT, Llop P, Penyalver R, and Cambra M (2003) Innovative tools for detection of plant pathogenic viruses and bacteria. Int Microbiol 6:233–43.

  3. Striebel HM, Birch-Hirschfeld E, Egerer R, and Foldes-Papp Z (2003) Virus diagnostics on microarrays. Curr Pharm Biotechnol 4:401–15.

  4. Lovmar L, Fredriksson M, Liljedahl U, Sigurdsson S, and Syvanen AC (2003) Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA. Nucleic Acids Res 31:e129.

  5. Wang D, Coscoy L, Zylberberg M, Avila PC, Boushey HA, Ganem D, and DeRisi JL (2002) Microarray-based detection and genotyping of viral pathogens. Proc Natl Acad Sci 99:15687–92.

  6. Wang D, Urisman A, Liu YT, Springer M, Ksiazek TG, Erdman DD, Mardis ER, Hickenbotham M, Magrini V, Eldred J, Latreille JP, Wilson RK, Ganem D, DeRisi JL (2003) Viral discovery and sequence recovery using DNA microarrays. PLoS Biol 1:257–60.