Experimental evaluation of the FluChip diagnostic microarray for influenza virus surveillance

Abstract

Global surveillance of influenza is critical for improvements in disease management and is especially important for early detection, rapid intervention, and a possible reduction of the impact of an influenza pandemic. Enhanced surveillance requires rapid, robust, and inexpensive analytical techniques capable of providing a detailed analysis of influenza virus strains. Low-density oligonucleotide microarrays with highly multiplexed "signatures" for influenza viruses offer many of the desired characteristics. However, the high mutability of the influenza virus represents a design challenge. In order for an influenza virus microarray to be of utility, it must provide information for a wide range of viral strains and lineages. The design and characterization of an influenza microarray, the FluChip-55 microarray, for the relatively rapid identification of influenza A virus subtypes H1N1, H3N2, and H5N1 are described here. In this work, a small set of sequences was carefully selected to exhibit broad coverage for the influenza A and B viruses currently circulating in the human population as well as the avian A/H5N1 virus that has become enzootic in poultry in Southeast Asia and that has recently spread to Europe. A complete assay involving extraction and amplification of the viral RNA was developed and tested. In a blind study of 72 influenza virus isolates, RNA from a wide range of influenza A and B viruses was amplified, hybridized, labeled with a fluorophore, and imaged. The entire analysis time was less than 12 h. The combined results for two assays provided the absolutely correct types and subtypes for an average of 72% of the isolates, the correct type and partially correct subtype information for 13% of the isolates, the correct type only for 10% of the isolates, false-negative signals for 4% of the isolates, and false-positive signals for 1% of the isolates. In the overwhelming majority of cases in which incomplete subtyping was observed, the failure was due to the nucleic acid amplification step rather than limitations in the microarray.

FIG. 1.

FluChip-55 layout. Capture sequences were spotted in triplicate next to positive control (PC) rows. Samples were grouped in columns by subtype (HA and NA) or by type (influenza A virus M [A M] or influenza B virus M [B M]). Sequences for influenza B virus NP and HA are grouped as well.

FIG. 2.

Typical microarray results demonstrating correct typing and subtyping of (a) A/H1N1, (b) A/H3N2, and (c) A/H5N1. The dark spots represent strong fluorescence signals. The spots at the top and left are positive controls, as detailed in the Materials and Methods section. The boxed areas highlight hits for specific subtypes, with the designations included for ease of viewing. For reference, the signal-to-noise values, defined as the signal minus the background divided by the standard deviation of the background, for the seven influenza A virus matrix protein sequences for A/H5N1 in panel c (denoted 1 to 7 in the image) are 35, 2, 60, 57, 3, 248, and 13, respectively. The typical relative variation in the signal for triplicate spots is 10%. The limit of detection on the microarray was ∼0.7 ng RNA.

FIG. 3.

Bar graph summary of results for analysis of 72 samples by using the assay (with influenza A virus-specific primers only) in conjunction with the FluChip-55 microarray. The overall assay performance, defined simply as the volunteer's success in identifying viruses based on our microarray images, is summarized for both the original blind study (a) and a duplicate study (b). Identifications were grouped according to complete and correct assignment (Complete), correct type and partial subtype (Partial), correct type only (Type Only), and false-negative (FN) or false-positive (FP) hits. The microarray performance, which has been corrected for missing subtypes and a lack of RNA amplification, is shown for the blind (c) and duplicate (d) studies.

FIG. 4.

An ethidium bromide-stained 1% agarose gel showing PCR products for several influenza virus samples. The amplified gene is noted on the right, while the fragment size is marked on the left.

FIG. 5.

Representative image showing the correct typing and subtyping of patient sample derived influenza A H3N2 virus.