BACKGROUND
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  and neuraminidase .
a total of  <dig> ha subtypes  and  <dig> na subtypes  have been identified.
many subtypes of influenza virus are found in aquatic birds, and some of them have been reported to infect humans  <cit> .
the two most common subtypes of influenza a virus currently circulating in humans are h1n <dig> and h3n <dig>  the recent pandemic influenza virus, a novel swine influenza a/h1n <dig> virus ), which was first isolated in mexico city in march  <dig>  <cit> , has spread to all states, and resulted in  <dig>  hospitalized cases and  <dig> deaths in the united states as of september  <dig>   <dig>  in addition, highly pathogenic avian influenza  virus h5n <dig> has spread throughout asia, europe, the middle east and the african continent, and was also documented to infect humans  <cit> .
h5n <dig> virus infection continues to progress with expanding host range and poses a serious global threat  <cit> .
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  and hemagglutinin inhibition immunoassays, and require 3- <dig> days  <cit> .
nucleic acid testing  of influenza viruses has been widely implemented over the last decade using target amplification methods such as reverse transcription-pcr , real-time rt-pcr, nucleic acid sequence-based amplification and loop-mediated isothermal amplification  <cit> .
these technologies are being employed for the rapid diagnosis of influenza a, in particular the subtyping of the h <dig> gene  <cit> .
for example, the taqman influenza a/h <dig> virus detection kit , a pcr-based method, was developed as an essential research and diagnostic tool for detection of a broad range of subtypes of influenza viruses  <cit> .
additionally, hoffmann et al.
described a rapid real-time rt-pcr test specific for the hpai virus h5n <dig>  without the need for sequencing  <cit> .
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  <cit> .
microarray test methods have proven to be powerful tools for viral identification and subtyping  <cit> .
for example, the fluchip microarray has been reported to detect h1n <dig>  h3n <dig> and h5n <dig> strains in less than  <dig> hours  <cit> .
the mchip microarray was able to identify influenza a virus with 95% sensitivity and 92% specificity  <cit> .
combimatrix corporation has also developed a semiconductor-based influenza a research microarray that can detect all known subtypes of influenza a viruses within  <dig> hours  <cit> .
more recently, a low-density microarray utilizing the nanochip <dig> system , which employs one probe for the conserved m gene and  <dig> probes for the cleavage site region of ha gene, was described to be a useful diagnostic tool for h5n <dig> virus  <cit> .
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  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  <cit> .
in recent years there has been increasing interest in the use of nanoparticles  coupled with silver staining for diagnostic applications  <cit>  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  in human genomic dna samples without the need for template amplification  <cit> .
in this report, we describe the development of a new np-based genomic microarray assay that specifically identifies h5n <dig> 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 h5n <dig> viral genomic rna is added directly to the slides for hybridization and can be detected using gold np probes.
RESULTS
probes for the np-based microarray assay
* 25-mer poly  tail added at 3' end of each intermediate oligonucleotides
** m gene degenerated capture oligos from h1n <dig>  h3n <dig> and h5n <dig>
a gold np was functionalized by pcrl <dig> oligo as a internal control probe.
bsequence from publication by zou   <cit> .
specificity and sensitivity of the genomic microarray assay
a typical np-based genomic microarray assay is performed by directly hybridizing purified viral rna to the capture oligos attached to the slide and then introducing intermediate oligos with a poly-a tail to form a sandwich complex .
the complex is then incubated with a poly dt-modified gold-np probe that can bind to the intermediate oligos and be stained with silver solution .
the enhanced light-scattering signal produced by the silver shell is detected by nanosphere verigene ® reader.
the detection sensitivity of the np-based microarray assay was first evaluated using m and h <dig> gene pcr amplicons generated from the h5n <dig> virus strain 'a/vietnam/1203/04' .
a dose-response model in the assay signal intensity was observed when the pcr products were diluted from  <dig> pm to  <dig> fm, although different patterns resulted between the m and h <dig> genes, and among various capture oligos of the m gene.
the m gene capture oligos m <dig> and m <dig> could detect at least  <dig> fm of pcr amplicon  while three of the four capture oligos of the h <dig> gene also detected  <dig> fm of the h <dig> gene pcr amplicon .
a third m gene capture oligo, m <dig>  and the h <dig> gene capture oligo, h <dig>  could only detect  <dig> pm of the target.
it is noteworthy that the sensitivity of the assay is based on measurement of a purified pcr amplicon which may be different from genomic viral rna .
the pcr fragments of the m, h <dig> and n <dig> genes of h5n <dig> virus  were hybridized separately or simultaneously to further evaluate the assay specificity .
specific signal was only generated in the areas printed with the corresponding gene-specific capture oligos .
no interference was observed when multiple targets and intermediate oligos were mixed and incubated simultaneously .
therefore, the use of a three-step capture and three-level hybridization procedure in the current assay proved to be specific for detection of m, h <dig> and n <dig> genes of h5n <dig>
direct detection of h5n <dig> viral rna
the next set of experiments focused on directly detecting viral rna in the absence of enzymatic target amplification using the gold np-based microarray assay .
the consensus m gene oligos within the microarray assay could detect the m gene from all three of the major subtypes of influenza a viruses  tested in our study.
in addition, the n <dig> gene was detected in both h1n <dig>  and h5n <dig>  viruses, and the h <dig> gene was only detected in the h5n <dig> virus.
no h <dig> or n <dig> signals were observed for h3n <dig> virus .
the results of  <dig> influenza a viruses tested for the m, h <dig> and n <dig> genes showed in table  <dig>  as a proof-of-concept experiment, these results indicated that our assay conditions and format permit the detection of different strains of h5n <dig> virus and were able to discriminate between the major subtypes of influenza a viruses h1n <dig>  h3n <dig> and h5n <dig>
*h5n <dig> strain obtained from cdc
due to the lack of well-characterized standards for influenza a virus rna, the detection sensitivity of the np-based microarray assay was evaluated by using two different sources of viral rna.
first, the well-quantified rna transcribed from h1n <dig> m gene plasmid was used in the experiments.
the assay detected approximately  <dig> copies of transcribed rna based on signals from the m gene capture oligos m <dig> and m <dig> .
second, viral rna extracted from the h5n <dig> strain a/vietnam/1203/ <dig> with quantified tcid <dig> units was tested.
without rt-pcr amplification, the assay detected  <dig> tcid <dig> units of viral rna per reaction with the specific h <dig>  n <dig>  and m gene capture oligos based on a s/co of > <dig> .
statistical analysis indicated that capture oligo n <dig> had the highest detection sensitivity for the a/vietnam/1203/ <dig>  strain.
in a parallel experiment, the np-based microarray assay showed  <dig> fold lower sensitivity compared with the taqman assay .
discussion
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  <cit> , 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, h <dig> and n <dig> genes of influenza a virus h5n <dig> and for differentiation of h5n <dig> from h1n <dig> and h3n <dig> 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,  <dig> samples can be tested in approximately  <dig>  hours.
it is faster and less labor-intensive than previously reported assays such as the fluchip  <cit> , mchip  <cit>  and nanochip  <dig>  <cit> , which usually require 5- <dig> 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.
<dig>  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  were designed to identify the major influenza a viruses  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 .
for example, a/vietnam/1203/ <dig>  strain hybridized well with the m <dig> and m <dig> capture oligos but not with m <dig> .
the alignment of the three capture sequences with the m gene sequence of a/vietnam/1203/ <dig> strain indicated that there were two and four degenerate nucleotide substitutions in m <dig> and m <dig>  respectively, but there were three degenerate nucleotide substitutions and eleven nucleotide mismatches in m <dig>  this may explain why m <dig> was not able to detect the a/vietnam/1203/ <dig> strain but performed well with other strains, including h1n <dig> , h3n <dig> , and certain h5n <dig> strains.
furthermore, the same oligo design strategy was used for subtyping the h <dig> and n <dig> genes, and similar results were observed.
for example, the n <dig> gene capture oligo n <dig> can consistently detect the majority of viral strains tested whereas other n <dig> gene capture oligos exhibit more variation in performance across strains.
nonetheless, capture oligo n <dig> performed better than n <dig> in some h5n <dig> 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 h <dig> gene was correctly identified in well-characterized h5n <dig> samples and the n <dig> gene was correctly identified in all h5n <dig> and h1n <dig> 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 h5n <dig> strains.
our current np-based genomic array could detect  <dig> tcid <dig> units of h5n <dig> virus per reaction, which is within the range of proposed detection limits for influenza diagnostics  <cit> .
CONCLUSIONS
a novel, pcr-free, np-based genomic microarray assay for subtyping influenza a virus h5n <dig> 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 h5n <dig> 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 h5n <dig> strains and clinical samples.
the specific capture oligos for targeting h <dig>  h <dig>  h <dig>  n <dig>  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  <dig> a are  <dig> % and  <dig> % distinct respectively, in amino acid sequence, from the  <dig> h1n <dig> strain  <cit> , it is possible to modify this new np-based genomic microarray using specific oligos for the  <dig> influenza a  virus or other emerging pandemic influenza strains to facilitate identification and differentiation of these viruses from other circulating influenza a viral strains.

 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 and neuraminidase. a total of ha subtypes and na subtypes have been identified. many subtypes of influenza virus are found in aquatic birds, and some of them have been reported to infect humans. the two most common subtypes of influenza a virus currently circulating in humans are h1n and h3n the recent pandemic influenza virus, a novel swine influenza a/h1n virus ), which was first isolated in mexico city in march, has spread to all states, and resulted in hospitalized cases and deaths in the united states as of september in addition, highly pathogenic avian influenza virus h5n has spread throughout asia, europe, the middle east and the african continent, and was also documented to infect humans. h5n virus infection continues to progress with expanding host range and poses a serious global threat. 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 and hemagglutinin inhibition immunoassays, and require 3- days. nucleic acid testing of influenza viruses has been widely implemented over the last decade using target amplification methods such as reverse transcription-pcr, real-time rt-pcr, nucleic acid sequence-based amplification and loop-mediated isothermal amplification. these technologies are being employed for the rapid diagnosis of influenza a, in particular the subtyping of the h gene. for example, the taqman influenza a/h virus detection kit, a pcr-based method, was developed as an essential research and diagnostic tool for detection of a broad range of subtypes of influenza viruses. additionally, hoffmann et al. described a rapid real-time rt-pcr test specific for the hpai virus h5n without the need for sequencing. 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. microarray test methods have proven to be powerful tools for viral identification and subtyping. for example, the fluchip microarray has been reported to detect h1n h3n and h5n strains in less than hours. the mchip microarray was able to identify influenza a virus with 95% sensitivity and 92% specificity. combimatrix corporation has also developed a semiconductor-based influenza a research microarray that can detect all known subtypes of influenza a viruses within hours. more recently, a low-density microarray utilizing the nanochip system, which employs one probe for the conserved m gene and probes for the cleavage site region of ha gene, was described to be a useful diagnostic tool for h5n virus. 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 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. in recent years there has been increasing interest in the use of nanoparticles coupled with silver staining for diagnostic applications 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 in human genomic dna samples without the need for template amplification. in this report, we describe the development of a new np-based genomic microarray assay that specifically identifies h5n 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 h5n viral genomic rna is added directly to the slides for hybridization and can be detected using gold np probes.  probes for the np-based microarray assay * 25-mer poly tail added at 3' end of each intermediate oligonucleotides ** m gene degenerated capture oligos from h1n h3n and h5n a gold np was functionalized by pcrl oligo as a internal control probe. bsequence from publication by zou. specificity and sensitivity of the genomic microarray assay a typical np-based genomic microarray assay is performed by directly hybridizing purified viral rna to the capture oligos attached to the slide and then introducing intermediate oligos with a poly-a tail to form a sandwich complex. the complex is then incubated with a poly dt-modified gold-np probe that can bind to the intermediate oligos and be stained with silver solution. the enhanced light-scattering signal produced by the silver shell is detected by nanosphere verigene ® reader. the detection sensitivity of the np-based microarray assay was first evaluated using m and h gene pcr amplicons generated from the h5n virus strain'a/vietnam/1203/04'. a dose-response model in the assay signal intensity was observed when the pcr products were diluted from pm to fm, although different patterns resulted between the m and h genes, and among various capture oligos of the m gene. the m gene capture oligos m and m could detect at least fm of pcr amplicon while three of the four capture oligos of the h gene also detected fm of the h gene pcr amplicon. a third m gene capture oligo, m and the h gene capture oligo, h could only detect pm of the target. it is noteworthy that the sensitivity of the assay is based on measurement of a purified pcr amplicon which may be different from genomic viral rna. the pcr fragments of the m, h and n genes of h5n virus were hybridized separately or simultaneously to further evaluate the assay specificity. specific signal was only generated in the areas printed with the corresponding gene-specific capture oligos. no interference was observed when multiple targets and intermediate oligos were mixed and incubated simultaneously. therefore, the use of a three-step capture and three-level hybridization procedure in the current assay proved to be specific for detection of m, h and n genes of h5n direct detection of h5n viral rna the next set of experiments focused on directly detecting viral rna in the absence of enzymatic target amplification using the gold np-based microarray assay. the consensus m gene oligos within the microarray assay could detect the m gene from all three of the major subtypes of influenza a viruses tested in our study. in addition, the n gene was detected in both h1n and h5n viruses, and the h gene was only detected in the h5n virus. no h or n signals were observed for h3n virus. the results of influenza a viruses tested for the m, h and n genes showed in table as a proof-of-concept experiment, these results indicated that our assay conditions and format permit the detection of different strains of h5n virus and were able to discriminate between the major subtypes of influenza a viruses h1n h3n and h5n *h5n strain obtained from cdc due to the lack of well-characterized standards for influenza a virus rna, the detection sensitivity of the np-based microarray assay was evaluated by using two different sources of viral rna. first, the well-quantified rna transcribed from h1n m gene plasmid was used in the experiments. the assay detected approximately copies of transcribed rna based on signals from the m gene capture oligos m and m. second, viral rna extracted from the h5n strain a/vietnam/1203/ with quantified tcid units was tested. without rt-pcr amplification, the assay detected tcid units of viral rna per reaction with the specific h n and m gene capture oligos based on a s/co of >. statistical analysis indicated that capture oligo n had the highest detection sensitivity for the a/vietnam/1203/ strain. in a parallel experiment, the np-based microarray assay showed fold lower sensitivity compared with the taqman assay. discussion 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, 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, h and n genes of influenza a virus h5n and for differentiation of h5n from h1n and h3n 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, samples can be tested in approximately hours. it is faster and less labor-intensive than previously reported assays such as the fluchip, mchip and nanochip, which usually require 5- 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. 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 were designed to identify the major influenza a viruses 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. for example, a/vietnam/1203/ strain hybridized well with the m and m capture oligos but not with m. the alignment of the three capture sequences with the m gene sequence of a/vietnam/1203/ strain indicated that there were two and four degenerate nucleotide substitutions in m and m respectively, but there were three degenerate nucleotide substitutions and eleven nucleotide mismatches in m this may explain why m was not able to detect the a/vietnam/1203/ strain but performed well with other strains, including h1n, h3n, and certain h5n strains. furthermore, the same oligo design strategy was used for subtyping the h and n genes, and similar results were observed. for example, the n gene capture oligo n can consistently detect the majority of viral strains tested whereas other n gene capture oligos exhibit more variation in performance across strains. nonetheless, capture oligo n performed better than n in some h5n 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 h gene was correctly identified in well-characterized h5n samples and the n gene was correctly identified in all h5n and h1n 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 h5n strains. our current np-based genomic array could detect tcid units of h5n virus per reaction, which is within the range of proposed detection limits for influenza diagnostics.  a novel, pcr-free, np-based genomic microarray assay for subtyping influenza a virus h5n 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 h5n 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 h5n strains and clinical samples. the specific capture oligos for targeting h h h n 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 a are % and % distinct respectively, in amino acid sequence, from the h1n strain, it is possible to modify this new np-based genomic microarray using specific oligos for the influenza a virus or other emerging pandemic influenza strains to facilitate identification and differentiation of these viruses from other circulating influenza a viral strains. 
viral rna was detected within hours using capture-target-intermediate oligonucleotide hybridization and gold np-mediated silver staining in the absence of rna fragmentation, target amplification, and enzymatic reactions. the lower limit of detection of the assay was less than fm for purified pcr fragments and tcid units for h5n viral rna. in this report, we describe a rapid and simple gold nanoparticle -based genomic microarray assay for specific identification of avian influenza virus h5n and its discrimination from other major influenza a virus strains. the new method described here may be useful for simultaneous detection and subtyping of major influenza a viruses. the np-based microarray assay was able to detect and distinguish h5n sequences from those of major influenza a viruses.
 for more than a decade there has been increasing interest in the use of nanotechnology and microarray platforms for diagnostic applications. in this report, we describe a rapid and simple gold nanoparticle -based genomic microarray assay for specific identification of avian influenza virus h5n and its discrimination from other major influenza a virus strains.  capture and intermediate oligonucleotides were designed based on the consensus sequences of the matrix gene of h1n h3n and h5n viruses, and sequences specific for the hemaglutinin and neuraminidase genes of the h5n virus. viral rna was detected within hours using capture-target-intermediate oligonucleotide hybridization and gold np-mediated silver staining in the absence of rna fragmentation, target amplification, and enzymatic reactions. the lower limit of detection of the assay was less than fm for purified pcr fragments and tcid units for h5n viral rna.  the np-based microarray assay was able to detect and distinguish h5n sequences from those of major influenza a viruses. the new method described here may be useful for simultaneous detection and subtyping of major influenza a viruses. 