Multiplexed, rapid detection of H5N1 using a PCR-free nanoparticle-based genomic microarray assay

Influenza A virus consists of eight negative single-stranded RNA segments and can be classified into various subtypes based on antigenic differences of two surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA). A total of 16 HA subtypes (H1-H16) and 9 NA subtypes (N1-N9) have been identified. Many subtypes of influenza virus are found in aquatic birds, and some of them have been reported to infect humans [1–5]. The two most common subtypes of influenza A virus currently circulating in humans are H1N1 and H3N2. The recent pandemic influenza virus, a novel swine influenza A/H1N1 virus (2009 A(H1N1)), which was first isolated in Mexico City in March 2009 [6], has spread to all states, and resulted in 9,079 hospitalized cases and 593 deaths in the United States as of September 11, 2009. In addition, highly pathogenic avian influenza (HPAI) virus H5N1 has spread throughout Asia, Europe, the Middle East and the African continent, and was also documented to infect humans [3, 7]. H5N1 virus infection continues to progress with expanding host range and poses a serious global threat [8]. Rapid and sensitive diagnostic tools for the identification of influenza viruses are crucial for early detection, appropriate treatment, epidemiologic investigations, and timely responses to a pandemic threat.

The most common methods for identification of influenza A viruses mainly depend on virus isolation, culture, characterization by polymerase chain reaction (PCR) and hemagglutinin inhibition immunoassays, and require 3-7 days [8–11]. Nucleic acid testing (NAT) of influenza viruses has been widely implemented over the last decade using target amplification methods such as reverse transcription-PCR (RT-PCR), real-time RT-PCR, nucleic acid sequence-based amplification and loop-mediated isothermal amplification [12–15]. These technologies are being employed for the rapid diagnosis of influenza A, in particular the subtyping of the H5 gene [14, 16–19]. For example, the Taqman influenza A/H5 Virus Detection Kit (Applied Biosystems, Foster City, CA), a PCR-based method, was developed as an essential research and diagnostic tool for detection of a broad range of subtypes of influenza viruses [20]. Additionally, Hoffmann et al. (2007) described a rapid real-time RT-PCR test specific for the HPAI virus H5N1 (Qinghai clade 2.2) without the need for sequencing [21]. These methods use universal primers and probes to detect all subtypes of influenza A, and specific primers and probes to distinguish HA and NA gene subtypes in a simple, single tube assay format following RNA isolation [13, 14, 18].

Microarray test methods have proven to be powerful tools for viral identification and subtyping [2, 22–25]. For example, the FluChip microarray has been reported to detect H1N1, H3N2 and H5N1 strains in less than 12 hours [22, 26]. The MChip microarray was able to identify influenza A virus with 95% sensitivity and 92% specificity [23, 27]. CombiMatrix Corporation has also developed a semiconductor-based Influenza A Research Microarray that can detect all known subtypes of influenza A viruses within 5 hours [28]. More recently, a low-density microarray utilizing the NanoChip400 system (Nanogen Inc), which employs one probe for the conserved M gene and 97 probes for the cleavage site region of HA gene, was described to be a useful diagnostic tool for H5N1 virus [29, 30]. However, all of these microarray based assays require two or more enzymatic amplification steps of influenza viral RNA prior to hybridization. In addition, detection requires labeling of multiple probes or incorporation of fluorescent dye- or biotin-conjugated nucleotides into double-stranded DNA (dsDNA) generated by RT-PCR. Moreover, the sensitivity of the conventional microarray assay relies on the efficiency of target amplification and hybridization of amplicons and probes. The multiple steps involved in these assays make them complicated, expensive, time consuming, susceptible to contamination, and may produce false negative results due to the presence of gene mutations, PCR inhibitors and RNA degradation. The design of multiple, specific primer sets and assay optimization pose major challenges [22].

In recent years there has been increasing interest in the use of nanoparticles (NP) coupled with silver staining for diagnostic applications [31–33] due to the higher sensitivities achievable by this approach compared with the fluorescent dyes that are commonly used in microarray assays. This modified microarray system allows direct detection of single nucleotide polymorphisms (SNPs) in human genomic DNA samples without the need for template amplification [33]. In this report, we describe the development of a new NP-based genomic microarray assay that specifically identifies H5N1 viral nucleic acid and simultaneously provides subtype identification of influenza A virus in the absence of target amplification procedures such as RT-PCR. The genomic microarray system has a high degree of hybridization efficiency and assay specificity. The method is also simple and rapid since the H5N1 viral genomic RNA is added directly to the slides for hybridization and can be detected using gold NP probes.

The conventional microarray assay requires target amplification by PCR and incorporation of a fluorescent dye- or biotin-conjugated nucleotide into PCR products prior to hybridization. These assays are both time- and labor-consuming and challenging to perform since multiple instruments are required for amplification and detection. Special precautions are needed to prevent carry-over contamination since detection of the resulting PCR amplicons is not performed in the same tube. In addition, RT-PCR may fail to amplify the target gene due to the highly variable sequences among influenza viruses [22], and multiple PCRs assays may be required to determine the subtypes of influenza A viruses. In this study, we developed a rapid, simple gold NP-based microarray assay for the simultaneous detection of M, H5 and N1 genes of influenza A virus H5N1 and for differentiation of H5N1 from H1N1 and H3N2 viruses. Our system combines specificity and capability for multiplexed detection on a simplified microarray platform, and may potentially be used in resource-limited areas for monitoring or screening of viral strains circulating during an influenza pandemic. The unique advantages of the new system include: 1) direct detection of viral RNA without involvement of target amplification or other enzymatic steps; 2) capture of a wide variety of strains by using multiple capture oligos that target different viral genes and sequences; 3) simultaneous detection and subtyping of major influenza A viruses; 4) a relatively simple, and rapid format. After RNA extraction, 20 samples can be tested in approximately 2.5 hours. It is faster and less labor-intensive than previously reported assays such as the FluChip [22, 26], MChip [23, 27] and NanoChip 400 [29, 30], which usually require 5-12 hours for detection.

Preliminary data indicate that the assay is able to achieve a high degree of sensitivity without PCR amplification. One reason is that the new detection method utilizes gold NP-based silver enhancement, which provides higher sensitivity than the traditional fluorescent labels. Another possible reason for higher detection sensitivity is the use of multiple capture and intermediate oligos to detect a target in multiple regions, which significantly increases the binding of gold NPs and could overcome the diagnostic challenge posed by potential gene mutations. The assay may also be suitable for point-of-care settings with further modifications of the detection systems. Indeed, the experiments we performed showed that the microarray signals could be visualized by the naked eye, without the need for a device, when sufficiently high viral RNA copies are present in the samples.

The genomic microarray assay involves three steps with three levels of capture-target-intermediate oligo hybridization as indicated in Fig. 1. Our results demonstrate that selection of target specific capture sequences is critical for performance of this assay, and the use of multiple capture and intermediate oligos is critical for detecting a wide variety of strains with the desired sensitivity. Three degenerate capture oligos for M gene (M01, M02 and M03) were designed to identify the major influenza A viruses (H1N1, H3N2 and H5N1) in our assay since the M gene is relatively well-conserved among influenza A viruses. Due to genetic variation of different virus strains, the performance of the different capture oligos varied (Fig. 2C and 2D). For example, A/Vietnam/1203/04 (H5N1) strain hybridized well with the M01 and M03 capture oligos but not with M02 (Fig. 2B and 2C). The alignment of the three capture sequences with the M gene sequence of A/Vietnam/1203/04 strain indicated that there were two and four degenerate nucleotide substitutions in M01 and M03, respectively, but there were three degenerate nucleotide substitutions and eleven nucleotide mismatches in M02. This may explain why M02 was not able to detect the A/Vietnam/1203/04 strain but performed well with other strains, including H1N1 (Fig. 2D, A/Hong Kong/29/06), H3N2 (A/Hong Kong/8/68), and certain H5N1 strains. Furthermore, the same oligo design strategy was used for subtyping the H5 and N1 genes, and similar results were observed. For example, the N1 gene capture oligo N104 can consistently detect the majority of viral strains tested whereas other N1 gene capture oligos exhibit more variation in performance across strains. Nonetheless, capture oligo N102 performed better than N104 in some H5N1 strains. These results suggest that multiple captures are necessary for improving detection sensitivity.

Using this multiple capture approach, the presence of influenza A viral RNA was verified in all seventeen strains of different influenza A viruses tested. The H5 gene was correctly identified in well-characterized H5N1 samples and the N1 gene was correctly identified in all H5N1 and H1N1 samples tested. No cross-reactivity was observed when other influenza A strains were tested. These results demonstrate the specificity and accuracy of our assay for detecting and subtyping H5N1 strains. Our current NP-based genomic array could detect 10³ TCID₅₀ units of H5N1 virus per reaction, which is within the range of proposed detection limits for influenza diagnostics [34].

A novel, PCR-free, NP-based genomic microarray assay for subtyping influenza A virus H5N1 was developed and evaluated using different influenza A strains. Preliminary data indicate that the method may be useful in surveillance and rapid identification of influenza A viral infection, particularly during H5N1 outbreaks. This proof-of-concept study provides evidence that the new system may be used to characterize multiple influenza A viruses during a pandemic and has the potential to simultaneously detect multiple, major influenza A viruses. We believe that assay sensitivity can be further improved through sample enrichment and optimization of assay conditions. Further studies are needed to validate the current method with additional H5N1 strains and clinical samples. The specific capture oligos for targeting H1, H3, H7, N2, and other genes in additional influenza A viruses will be generated in the future to expand the current identification panel. Thus, this new NP-based genomic array system provides the flexibility to be rapidly modified to detect new targets. Since the viral HA and NA genes of the recent outbreak of swine influenza 2009 A(H1N1) are 27.2% and 18.2% distinct respectively, in amino acid sequence, from the 2008 H1N1 strain [35], it is possible to modify this new NP-based genomic microarray using specific oligos for the 2009 influenza A (H1N1) virus or other emerging pandemic influenza strains to facilitate identification and differentiation of these viruses from other circulating influenza A viral strains.