Typing and Subtyping Influenza Virus Using DNA Microarrays and Multiplex Reverse Transcriptase PCR

Abstract

A model DNA microarray has been prepared and shown to facilitate typing and subtyping of human influenza A and B viruses. Reverse transcriptase PCR was used to prepare cDNAs encoding ϳ500-bp influenza virus gene fragments, which were then cloned, sequenced, reamplified, and spotted to form a glass-bound microarray. These target DNAs included multiple fragments of the hemagglutinin, neuraminidase, and matrix protein genes. Cy3-or Cy5-labeled fluorescent probes were then hybridized to these target DNAs, and the arrays were scanned to determine the probe binding site(s). The hybridization pattern agreed perfectly with the known grid location of each target, and the signal-to-background ratio varied from 5 to 30. No crosshybridization could be detected beyond that expected from the limited degree of sequence overlap between different probes and targets. At least 100 to 150 bp of homology was required for hybridization under the conditions used in this study. Combinations of Cy3-and Cy5-labeled DNAs can also be hybridized to the same chip, permitting further differentiation of amplified molecules in complex mixtures. In a more realistic test of the technology, several sets of multiplex PCR primers that collectively target influenza A and B virus strains were identified and were used to type and subtype several previously unsequenced influenza virus isolates. The results show that DNA microarray technology provides a useful supplement to PCR-based diagnostic methods.

Swine Outbreak of Pandemic Influenza A Virus on a Canadian Research Farm Supports Human-to-Swine Transmission

“…Finally, molecular characterization studies showed that no molecular adaptive changes occurred and almost identical viruses infected humans and swine. There were only minor differences between human and swine pH1N1 isolates, which is likely, given the natural mutation rate of influenza virus both in vitro and in vivo [16,17].…”

Impact of Microarray Technology in Influenza Virus Research and Diagnostics

“…Microarrays are the new diagnostics tools for identification and quick subtyping of influenza viruses. For this low-density oligonucleotide microarrays containing limited number of highly multiplexed unique conserved genome "signatures" sequences as probes are used [9][10][11][12][13]. These microarrays are used to detect both influenza A and B viruses as well as various subtypes of influenza A viruses on the basis of three gene targets the HA, NA and M (Matrix) gene segments [6,10,14,15].…”

“…The study could identify 3 unknown influenza viruses; however the technique suffered from high degree of mismatches (approx. 14%) [13]. In order to resolve this problem smaller oligonucleotide based arrays were designed containing conserved viral genome for greater specificity.…”

“…These microarrays use sets of immobilized oligonucleotide probes that form complexes with virus specific cDNA tagged with, a fluorescent label [8,11,12]. These microchips are used for the determination of different influenza virus types [13] and also for sub-typing majority of HA and NA influenza virus variants [21]. Hybridization microchip contains sets of HA (H1-H13, H15 and H16) and NA (N1-N9) oligonucleotide probes to be classified into different HA-NA subtypes of influenza viruses.…”

Detection and Differentiation of Four Poultry Diseases Using Asymmetric Reverse Transcription Polymerase Chain Reaction in Combination with Oligonucleotide Microarrays

“…The PCR method was used to amplify and label the reference gene. 13 This reaction mixture (total volume: 100 ml) consisted of 10 ml of 103 PCR buffer, 4 ml of dNTP (mixed with Cy3-29-deoxyuridine 59-triphosphate [dUTP]), j 1 ml of Taq DNA recombinant polymerase (rTaq), k 2 ml of forward primer, 2 ml of Cy3-labeled reverse primer, 1 ml of GAPDH plasmid, and 80 ml of DEPC-treated sterile double distilled H 2 O. The thermal cycler conditions were 5 min of predenaturation at 95uC, followed by 35 cycles of 94uC for 45 sec, 53uC for 45 sec, and 72uC for 60 sec; and a final 10-min extension at 72uC.…”