Microarray-based detection and genotyping of viral pathogens

Abstract

The detection of viral pathogens is of critical importance in biology, medicine, and agriculture. Unfortunately, existing techniques to screen for a broad spectrum of viruses suffer from severe limitations. To facilitate the comprehensive and unbiased analysis of viral prevalence in a given biological setting, we have developed a genomic strategy for highly parallel viral screening. The cornerstone of this approach is a long oligonucleotide (70-mer) DNA microarray capable of simultaneously detecting hundreds of viruses. Using virally infected cell cultures, we were able to efficiently detect and identify many diverse viruses. Related viral serotypes could be distinguished by the unique pattern of hybridization generated by each virus. Furthermore, by selecting microarray elements derived from highly conserved regions within viral families, individual viruses that were not explicitly represented on the microarray were still detected, raising the possibility that this approach could be used for virus discovery. Finally, by using a random PCR amplification strategy in conjunction with the microarray, we were able to detect multiple viruses in human respiratory specimens without the use of sequence-specific or degenerate primers. This method is versatile and greatly expands the spectrum of detectable viruses in a single assay while simultaneously providing the capability to discriminate among viral subtypes.

Microfluidic Quantitative PCR for Simultaneous Quantification of Multiple Viruses in Environmental Water Samples

“…Pathogen concentrations obtained with this system could then be used for quantitative microbial risk assessment (QMRA) (19). Several advantages of this MFQPCR over other simultaneous multipathogen detection technologies such as microarray (20,21), TaqMan array (22,23), Luminex assay (24), OpenArray (25), FilmArray (26), and molecular inversion probe assay (27) include its high sensitivity and quantitative performance. However, MFQPCR technology has not been applied to quantify multiple viral pathogens.…”

PhyloFlu, a DNA Microarray for Determining the Phylogenetic Origin of Influenza A Virus Gene Segments and the Genomic Fingerprint of Viral Strains

“…Several studies have reported the use of DNA microarrays for the detection or classification of influenza viruses (26)(27)(28)(29)(30)(31). These microarrays were designed to detect specific influenza A virus strains (29,30) or to subtype and pathotype the HA of avian influenza viruses (30) but not to determine both the HA and NA subtypes.…”

Detection of Adventitious Viruses from Biologicals Using a Broad-Spectrum Microbial Detection Array

“…In designing probes for our array, we sought to balance the goals of conservation and uniqueness, prioritizing oligo sequences that were conserved, to the extent possible, within the family of the targeted organism, and unique relative to other families and kingdoms. We designed arrays with larger numbers of probes per sequence (50 or more for viruses, 15 or more for bacteria) than previous arrays having only [2][3][4][5][6][7][8][9][10] probes per target (5,6). The large number of probes per target was expected to improve sensitivity, an important consideration given possible amplification bias in the random PCR sample preparation protocol, which could result in non-amplification of genome regions targeted by some probes.…”