﻿JOURNAL OF CLINICAL MICROBIOLOGY,
0095-1137/01/$04.000 DOI: 10.1128/JCM.39.2.696­704.2001
Feb. 2001, p. 696­704 Vol. 39, No. 2
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Typing and Subtyping Influenza Virus Using DNA Microarrays
and Multiplex Reverse Transcriptase PCR
JIPING LI,1,2
SHU CHEN,2
AND DAVID H. EVANS1
*
Department of Molecular Biology and Genetics1
and Laboratory Services Division,2
The University of Guelph, Guelph, Ontario N1G 2W1, Canada
Received 21 September 2000/Accepted 15 November 2000
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 500-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 cross-
hybridization 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.
After the first reports describing DNA microchip arrays ap-
peared (15, 16), microarray technology revolutionized the
study of gene expression patterns in diverse organisms (re-
viewed in references 3 and 7). Arrays composed of oligonucle-
otides (15) or robotically spotted DNAs (16) permit genome
scale analysis of gene expression patterns and have more re-
cently been used in such applications as drug discovery (6),
mutation detection (8, 9, 12), evolutionary studies (11, 21), and
genome mapping (20). The application of DNA microarray
technology as a diagnostic tool also shows great promise, since
microarrays theoretically permit a simultaneous screen for any
of tens of thousands of nucleic acid sequences. However, only
recently have reports of these array-based applications started
to appear. Oligonucleotide arrays have been used to search for
mutations in cancer-linked genes (reviewed in reference 10)
and human immunodeficiency virus mutations (13, 25) and in
bacterial typing (1), but the high cost and limited availability of
such tools continue to limit research in this area.
We have been interested in the development of new tech-
nologies capable of better identifying viral pathogens and have
been using, as a model system, human influenza viruses (28,
29). Influenza viruses cause annually recurrent epidemics of
moderate-to-severe respiratory disease, frequently associated
with genetic variation termed drift and shift (5, 27). These
viruses present an important diagnostic problem, and the rapid
detection, typing, and subtyping of influenza A and B virus
strains are of both clinical and epidemiological value. While
antibody-based methods still form the foundations of routine
diagnostic work, many reports over the last decade have dem-
onstrated the utility and superiority of PCR-based diagnostic
(2, 4, 14, 19, 22, 26) and retrospective (23) tests. Unfortunately
PCR-based methods suffer from a problem in that simply pro-
ducing DNA isn't sufficient evidence that one has amplified the
right product. Other methods such as DNA sequencing (29),
blotting (18), and fluorogenic PCR (19) are required if one
desires proof that a PCR has amplified a bona fide nucleic acid
target. While such confirmatory methods are certainly reliable,
they still present something of a financial, technical, and logis-
tical challenge to busy laboratories routinely screening for hun-
dreds of different agents. They also become more difficult to
apply when multiplex methods are being used to simulta-
neously amplify two or more PCR products from a mixture of
templates.
DNA arrays offer a potential solution to these problems.
The arrays can potentially encode tens of thousands of possible
target sequences and thus provide a simple way of storing and
indexing numerous hybridization probes. In principle one
could draw this resource when needed to confirm the identity
of one or more PCR products by hybridization, with only
minimal modification to existing PCR-based protocols. We
have tested this approach and show here that a model DNA
array can be used to type and subtype influenza A and B virus
strains. This shows that DNA arrays can provide multiply re-
dundant confirmatory evidence that a PCR product encodes
the sequence(s) it is expected to encode.
MATERIALS AND METHODS
Viruses. Human influenza virus strains (Table 1) were obtained from the
American Type Culture Collection (Manassas, Va.) and propagated where nec-
essary in 10-day-old embryonated chicken eggs (29). Crude viral RNA was
extracted as described previously (29) and stored at 70°C.
Oligonucleotide primer design. Oligonucleotide primers were designed using
RightPrimer, version 1.2, software and GenBank Blastn sequence alignments.
Selected PCR primers meet the following criteria: (i) primers hybridize to highly
* Corresponding author. Mailing address: Department of Molecular
Biology and Genetics, University of Guelph, Guelph, Ontario N1G
2W1, Canada. Phone: (519) 824-4120, ext. 2575. Fax: (519) 837-2075.
E-mail: dhevans@uoguelph.ca.
696
conserved sequence elements, (ii) each amplicon spans approximately 500 bp,
(iii) amplified segments collectively encompass an entire gene (thus 1.5 kb of
virus DNA sequence required three primer pairs), and (iv) primer melting points
generally fell between 50 and 54°C. Desalted primers were purchased from
Gibco/BRL and used without further purification.
RT-PCR and cDNA cloning. A commercial kit was used to prepare viral
cDNAs. Reaction mixtures contained 10 pg of RNA, 200 M (each) de-
oxynucleoside triphosphate, 1.5 mM MgCl2, 0.4 M (each) primer, and other
components as directed by the manufacturer (Titan one-tube reverse transcrip-
tase PCR [RT-PCR] kit [Roche]). Reaction mixtures were incubated at 50°C for
30 min, denatured at 94°C for 3 min, and then subjected to 35 thermal cycles
(94°C for 30 s, 45°C for 30 s, 68°C for 2 min). Following a final 10-min incubation
at 68°C, the samples were chilled and the products were sized by electrophoresis.
PCR-amplified viral cDNAs were subsequently cloned using a Topo TA cloning
kit (Invitrogen). Recombinant plasmids were purified from lacZ mutant bacteria
and sequenced as described previously (24).
Preparation of target DNAs. M13 forward (5 GTAAAACGACGGCCAGTG
3) and reverse (5 CAGGAAACAGCTATGACC 3) primers were used to
reamplify cloned viral cDNAs. The reverse primer incorporated an amine tag
linked by a six-carbon spacer to the 5 end (Gibco/BRL). PCR mixtures (100 l)
contained 2 mM MgCl2, 200 M (each) deoxynucleoside triphosphate, 0.4 M
(each) primer, 1 ng of purified plasmid DNA, 1/10-diluted enzyme buffer, and
1.5 U of Taq polymerase (Perkin-Elmer). Following 35 thermal cycles (typically
94°C for 30 s, 52°C for 30 s, 72°C for 1 min) the DNA was purified using
MicroSpin S-400 columns (Amersham/Pharmacia Biotech), precipitated with
ethanol, resuspended in 30 l of 3 SSC (10 SSC is 87.6 g of NaCl/liter and
44.1 of sodium citrate/liter, pH 7.0), and the DNA concentration was adjusted to
300 ng/l for spotting purposes.
Microarray printing and processing. Amine-tagged target DNAs were distrib-
uted, in duplicate, into 384-well microtiter plates. A custom-built arrayer
(Virtek) was used to spot DNA on aldehyde-activated silylated microscope slides
(CEL Associates). Printed arrays were air dried for a few minutes at 50 to 60°C
and then stored overnight at 20 to 37°C over desiccant. The arrays were rehy-
drated for 4 h in a humid atmosphere, dried briefly at 50°C on a heating block,
washed once in 0.2% sodium dodecyl sulfate (SDS) and twice in water (1 min
each), and treated with sodium borohydride (1.0 g of NaBH4 [Sigma] dissolved
in 300 ml of phosphate-buffered saline plus 100 ml of ethanol [17]) for 5 min. The
DNA was denatured in water (2 min at 95°C) and then washed again (once in
0.2% SDS and once in water [1 min each]). Arrays were air dried and stored at
room temperature.
Probe preparation and multiplex PCR. Two methods for incorporating fluoro-
linked Cy3- and/or Cy5-dCTP (Amersham/Pharmacia Biotech) into fluorescent
probes were devised. The simplest method involved adding 20 M Cy3- or Cy5-
dCTP to a standard 100-l PCR mixture containing Taq polymerase; 200 M
(each) dATP, dGTP, and TTP plus 100 M dCTP; and a single primer pair (see
above). In subsequent experiments, probes were prepared using multiplex RT-
PCR mixtures also supplemented with 20 M Cy3- or Cy5-dCTP. In this case the
primers were combined into three different groups, but in such a way that the
combination of primers ensured that one influenza virus subtype could be am-
plified no matter which viral RNA or cloned cDNA was present. Labeled probes
were purified using MicroSpin S-300 columns and heat denatured before hybrid-
ization. Fluorescent molecules were handled under dim lighting to minimize
photobleaching.
Hybridization and data analysis. Microarrays were prehybridized in 20 l of
DIG Easy Hyb (Roche) containing 5 g of denatured salmon sperm DNA at
62°C for 1 h under a 12- by 12-mm coverslip. The coverslip was washed off the
slide in 0.1 SSC, and the slides were dried at room temperature. A fresh
solution of DIG Easy Hyb, containing 5 l of denatured fluorescent probe plus
5 g of salmon sperm DNA in a total volume of 20 l, was then applied to the
array and overlaid with a 12- by 12-mm coverslip. The arrays were incubated
overnight at 58 to 62°C in a humid chamber and then washed for 5 min at 20°C
in 1 SSC­0.1% SDS followed by 0.1 SSC­0.1% SDS. The arrays were rinsed
in 0.1 SSC, dried, and stored in the dark. Arrays were analyzed using GenePix
(Axon Instruments) or ChipReader (Virtek) confocal scanners, and the fluores-
cence was quantitated using ImaGene software (Biodiscovery). Gain settings
produced a linear detector response. A "glass" background fluorescence reading
was measured in the region surrounding each spot and subtracted, and the
intensity was normalized to produce an average fluorescence reading at all
nonhomologous spot positions equal to 50 U. The signal-to-background ratios
reported here are the fluorescence intensities measured at homologous spots
divided by an average measured at all other array locations.
Nucleotide sequence accession numbers. Viral sequences obtained in this
study have been assigned GenBank accession no. AF305216 to AF305220.
RESULTS
Viral genetic targets. Three types of human influenza viruses
are commonly encountered (A, B, and C), of which the A and
B types are of primary clinical interest. Type A strains are
further subtyped as encoding one of three different hemagglu-
tinins (HAs; H1, H2, or H3) and one of two different neura-
minidases (N1 or N2). The HA and neuraminidase (NA) genes
are principal pathogenic determinants that reside on separate
subgenomic segments, and it is genetic reassortment of these
segments which creates the six primary human influenza A
virus subtypes which change over decades. Human type B
TABLE 1. Human influenza viruses used in this study
Virus Subtype
GenBank accession no. of virus gene: DNAa
HA NA MP Arrayed Hybridized
A/New Jersey/8/76 H1N1 n/ab
M27970 n/a HA1, NA1
A/Denver/1/57 H1N1 AF305218c
AF305216c
AF305217c
HA1, NA1, MP
A/Japan/305/57 H2N2 L20406 n/a n/a HA2, NA2-3
A/Victoria/3/75 H3N2 V01098 J02173 n/a HA3 HA3, NA2, MP
A/Port Chalmers/1/73 H3N2 AF092062 n/a X08092 NA2-1, NA2-2, MP
B/Maryland/1/59 K00424 M30633 n/a HA, NA, MP
B/Hong Kong/5/72 AF305219c
AF305220c
n/a HA, NA, MP
a
Unless otherwise indicated, all DNA subfragments derived from the indicated virus. The one exception was NA2 gene fragments which derived from two different
viruses.
b
n/a, not available by April 2000.
c
Not available by April 2000. A partial sequence was determined in this study.
TABLE 2. Virus sequences used in primer design
Virus gene
Representative
strain
Accession
no.
Sequence
identity (%)a
Influenza A virus
HA-1 A/Kiev/59/79 M38353 89­98
HA-2 A/Singapore/1/57 L20410 95­99
HA-3 A/Aichi/2/68 J02090 87­99
NA-1 A/WS/33 L25816 81­99
NA-2 A/Victoria/3/75 J02173 92­99
MP A/PR/8/34 V01099 89­100
Influenza B virus
HA B/Singapore/222/79 X00897 95­99
NA B/Hong Kong/8/73 M30631 93­98
MP B/Lee/40 J02094 91­94
a
Degree of nucleotide sequence variation compared with other influenza gene
homologs in the NCBI database.
VOL. 39, 2001 DNA ARRAY DETECTION OF INFLUENZA VIRUSES 697
FIG. 1. Identification of influenza cDNAs using glass-supported microarrays. DNA targets were spotted in duplicate in an eight-by-eight grid,
and bound probes were detected using confocal fluorescence microscopy. The hybridization probe or probes used in each experiment are indicated
below each image, and the array pattern is shown in Table 4. The images seen in panels 1 to 6 were obtained using short probes spanning mostly
a single target (see text for a further discussion of this point), while panel 7 shows an array hybridized to a longer cDNA fragment spanning three
contiguous targets (A/HA3-1, A/HA3-2, and A/HA3-3). The single array shown in panels 8 and 9 was hybridized to a mixture of Cy3-labeled
698 LI ET AL. J. CLIN. MICROBIOL.
influenza virus strains also encode HA and NA genes, but only
a single major genetic variant of each is commonly encoun-
tered. Thus the typing and subtyping of influenza A and B virus
strains require an ability to detect at least four HA and three
NA genes plus their drifted allelic variants. Other viral genes
are also useful in differentiating influenza A and B virus strains
and offer the advantage of being more genetically stable. In
this regard we have previously used the influenza matrix pro-
tein (MP) gene as a PCR target. Methods capable of differen-
tiating a total of four HA, three NA, and two MP gene targets
thus provide the capacity to type and subtype human influenza
A and B virus strains with some degree of redundancy.
Preparation of cloned virus cDNAs. We initially cloned mul-
tiple separate fragments of genes for three influenza A virus
HAs (A/HA1, A/HA2, and A/HA3), two influenza A virus NAs
(A/NA1 and A/NA2), and an influenza A virus MP (A/MP) as
well as an influenza B virus HA (B/HA), NA (B/NA), and MP
(B/MP). The nine genes each spanned 1 to 1.5 kb and have
little or no sequence homology. Primers were selected using a
combination of primer design software and Blastn sequence
alignments with the intent of locating each primer pair in
maximally conserved sequence regions spaced about 500 bases
apart. Table 2 lists the nine DNA sequences used to initiate
homology searches and to design primers, and Table 3 shows
the 52 primers generated by this approach. Based on available
sequence data, the variation in nucleotide sequence within
each influenza gene family ranges from 81 to 100% sequence
identity (Table 2). These 26 primer pairs were used in RT-
PCRs to amplify genes encoded by five different influenza virus
strains (Table 1). We obtained 24 of 26 possible cDNA prod-
ucts (the A/HA1-2 and B/MP-2 primer sets did not work),
cloned these cDNAs, and verified the DNA sequences.
Array fabrication. The 24 sequence-validated cDNA clones
were reamplified using M13 universal primers, in the process
adding a 5 amino tag to the reverse primer to permit covalent
attachment to a modified glass support. A custom-built Stan-
ford-type DNA arrayer was used to spot DNA, in duplicate,
onto activated silylated glass slides at densities of 1,100 or
2,500 spots/cm2
(300- or 200-m spacing, respectively). The
array pattern is shown in Table 4. In addition to virus cDNAs
we added control spots composed of Escherichia coli DNA and
3 SSC buffer. Several replicate sets of the eight-by-eight grid
were also printed elsewhere on the slide to facilitate statistical
analysis of hybridization signals.
Hybridization of arrays with cloned cDNAs. To determine
whether these arrayed cDNA targets retained their expected
hybridization properties, we first tested whether fluorescent
probes derived from the original cloned cDNA templates
would hybridize to a cognate spot(s). Standard PCR mixtures
were supplemented with Cy3-dCTP and used to reamplify each
of the cloned cDNAs. These fluorescent probes were purified
and individually hybridized to separate arrays, and then the
hybridization signals were analyzed using Virtek or Axon chip
readers. A selection of the resulting fluorescence images are
shown in Fig. 1 (panels 1 to 6). These can be decoded by
noting, for example, that a 520-bp A/HA3-1 probe (Table 3)
produced strong hybridization signals at duplicate spots lo-
cated in row 3, columns 1 and 2 (Fig. 1, panel 2). Overall,
duplicate hybridization signals were clearly detected at all of
the positions known to encode homologous DNA targets and
little cross-hybridization to unrelated spots or background flu-
orescence was detected with these supports.
The digital images generated by fluorescence scanners can
be very misleading if judged just by eye, because the appear-
ance varies greatly in response to changes in instrument set-
tings, background subtraction, and postacquisition gain factors.
Therefore we also measured the hybridization signal intensities
and compared the difference in fluorescence intensity between
spots encoding homologous versus nonhomologous targets.
(This ratio of fluorescence values served as a way of normal-
izing data across many different arrays, with the cross-hybrid-
ization background always assigned an arbitrary value of 50
fluorescence units.) Initial experiments detected approximately
threefold variations in signal intensity, which seemed to corre-
late with changes in spot size (data not shown). Furthermore,
arrays prepared early in the production cycle produced the
most-intense signals, while later batches of slides, encoding
smaller spots, produced weaker fluorescence signals. This ef-
fect was caused by the arraying pins blunting with use, causing
the spots to decrease in radius from 75 to 45 m. This
should reduce the quantity of bound DNA approximately
threefold (75/45
2
), which agreed well with the observed variation
in fluorescence intensities. Figure 2 shows a representative
selection of results subsequently obtained from 10 separate
hybridization experiments using arrays bearing primarily
45-m (radius) spots. The signal intensity still varied in these
experiments from target to target, ranging from 180 to 1,090
arbitrary fluorescence units, but the fluorescence intensities
measured at sites encoding homologous target sequences were
always significantly greater (at least threefold) than the signals
detected elsewhere on the array at nonhomologous targets.
Several additional factors probably contribute to the residual
variation, including variations in the concentrations and spe-
cific activities of fluorescent probes. However, a significant
confounding factor appeared to be the size of the target DNA.
Effect of target length on signal intensity. The primers used
in this study were designed to create some overlap between the
two or three target cDNAs derived from any given influenza
virus gene. This made it possible to estimate what minimal
target sequence length might be required to produce a hybrid-
ization signal. For example, targets A/HA2-1 and A/HA2-2
have 202 bp of common sequence and, by hybridizing a probe
derived from A/HA2-1 to these arrays, one can measure the
relative efficiency of probe binding to target A/HA2-1 (513
homologous base pairs) or to A/HA2-2 (202 homologous base
pairs) (Fig. 1, panels 8 and 10). The results derived from this
analysis are shown in Fig. 3. Generally it was noted that over-
laps of less than 100 bp produced poor or no hybridization
A/HA2-1 probes and Cy5-labeled B/NA-1 probes and scanned simultaneously for Cy3 (panel 8) and Cy5 (panel 9) dyes at excitation wavelengths
of 543 and 635 nm, respectively. For comparison, panel 10 shows another array hybridized to a mixture of Cy3-labeled A/HA2-1 and B/NA-1
probes. Panels 11 and 12 illustrate the use of multiplex RT-PCR and the ability to detect hybridization of heterologous A/Denver/1/57 and B/Hong
Kong/5/72 probes to A/Port Chalmers/1/73 and B/Maryland/1/59 targets.
VOL. 39, 2001 DNA ARRAY DETECTION OF INFLUENZA VIRUSES 699
signals, suggesting that this represents the minimal length of
target required under these conditions. In contrast, overlap-
ping sequences in excess of 100 bp produced readily detect-
able fluorescence signals and the intensity appeared roughly
dependent on the length of the overlapping region common to
target and probe.
To further test what effect longer targets have on signal
intensity, we took advantage of the fact that other primer
combinations can be used to produce a cDNA fragment span-
ning two or more target DNAs in a single RT-PCR. For ex-
ample, a reaction with primers 15 and 20 (Table 3) generates
a 1,607-bp probe fragment spanning targets A/HA3-1 (520 bp),
A/HA3-2 (444 bp), and A/HA3-3 (707 bp). This method of
probe preparation ensured that differences in the concentra-
tions and specific activities of probes weren't responsible for
variations in hybridization efficiency. As expected, this now
uniformly labeled DNA hybridized to all three homologous
targets on a single array (Fig. 1, panel 7), and again no signif-
icant cross-hybridization was detected. Quantitation of the flu-
orescence signals showed the relative intensity ratios to be 1.2
(A/HA3-1) to 1.0 (A/HA3-2) to 2.1 (A/HA3-3), which follows
the same trend as the relative size ratios (1.2 [A/HA3-1] to 1.0
[A/HA3-2] to 1.6 [A/HA3-3]). Therefore, as with traditional
blotting technologies, DNA arrays produce hybridization sig-
nals which are roughly proportional to target size.
Hybridization using mixed probes and multiple dyes. A dou-
ble-labeling experiment was also conducted to further test the
discriminatory capacity of these arrays. Control experiments
showed that a mixture of Cy3-labeled A/HA2-1 and B/NA-1
probes hybridized to the expected A/HA2-1 and B/NA-1 tar-
gets in a single array (Fig. 1, panel 10) producing fluorescent
spots of comparable intensities. No cross-hybridization signals
were detected beyond some additional binding to the A/HA2-2
locus (the A/HA2-1 probe has 202-bp homology with an
A/HA2-2 target). We then used different fluorescent dyes to
prepare A/HA2-1 probes labeled with Cy3-dCTP and B/NA-1
probes labeled with Cy5-dCTP. The probes were mixed to-
gether in equal amounts and hybridized to the array, and the
bound fluorescent probes were then detected using 543 (Cy3)
and 635 nm (Cy5) as the excitation wavelengths. The two
resulting images are shown in Fig. 1, panels 8 and 9. Cy3- and
Cy5-labeled probes seemed to hybridize with comparable effi-
ciencies, producing fluorescence signals of comparable inten-
sities. Moreover, the Cy3-labeled A/HA2-1 probes hybridized
only to homologous A/HA2-1 and (overlapping) A/HA2-2 tar-
gets and the Cy-5-labeled B/NA-1 probe hybridized to the
B/NA-1 target. These data show that one can differentiate a
mixture of probes prepared using separate dyes and thus derive
additional information regarding the probe composition.
Multiplex PCR. The preceding experiments showed that
influenza virus cDNA arrays can be used to accurately identify
different model probes either singly or in mixtures. However,
the eventual goal of these experiments is to improve on mul-
tiplex methods capable of typing and subtyping unknown vi-
ruses. We thus screened our primers for primer combinations
that are compatible in multiplex RT-PCRs. In recognition of
the fact that some gene combinations are mutually exclusive
(e.g., a virus is either H1 or H2 but can't be both), primer
combinations that should produce a single diagnostically infor-
mative PCR product in a reaction with any given type or
subtype of influenza virus were chosen. After testing large
numbers of primer combinations against a battery of cDNAs
templates, we eventually identified three multiplex primer
combinations that collectively type and subtype influenza
strains. These primer sets are summarized in Table 5. Figure 4
shows how these multiplex primer combinations can selectively
amplify a particular gene target in reactions with one of the
primer mixtures and a particular cloned cDNA target. For
example, primer mixture A (Table 5) amplified an 510-bp
A/HA1-1 product in a PCR with a cloned A/HA1-1 template
(Fig. 4, lane b) as well as 490-bp A/HA2-3, 700-bp
A/HA3-3, and 530-bp B/MP-1 products in PCRs with
A/HA2-3, A/HA3-3, and B/MP-1 templates, respectively (Fig.
4, lanes e, h, and k).
Typing and subtyping influenza viruses by RT-PCR and
array hybridization. In a now more realistic test of this method
FIG. 2. Quantitation of hybridization signals. A glass background
value was automatically subtracted from all measured fluorescence
intensities, and the mean intensity and standard deviation (n  4) were
then calculated for each spot. To permit comparison between different
arrays and probes, the average nonspecific cross-hybridization signal
detemined at nonhomologous targets was assigned an arbitrary value
of 50 fluorescence units. In all cases, a clear hybridization signal was
readily differentiated from this nonspecific background.
FIG. 3. Hybridization signals depend on the length of homology.
Hybridization signals were measured using a variety of probes and
targets, and the fluorescence intensity was plotted as a function of the
number of base pairs common to each probe and target. Positive
hybridization signals were significantly differentiated from a nonspe-
cific cross-hybridization background when the amount of base overlap
exceeded 100 to 150 bp.
700 LI ET AL. J. CLIN. MICROBIOL.
we examined whether the multiplex primers described in Table
5 can actually type and subtype different influenza viruses.
Three viral RNA strains were tested (A/Denver/1/57 [H1N1],
A/Victoria/3/75 [H3N2], and B/Hong Kong/5/72), with A/Den-
ver/1/57 and B/Hong Kong/5/72 being different from those
strains used to prepare arrayed targets (Table 1). None of the
sequences targeted in strains A/Denver/1/57 and B/Hong
Kong/5/72 were known to us when the work started, nor was
the sequence of the MP gene of A/Victoria/3/75. Multiplex
RT-PCR succeeded in producing all six of the six possible
probes from mixtures containing influenza A virus RNA
(HA1-1, HA3-3, NA1-1, NA2-2, and two MP-1 gene frag-
ments) and two of the three possible probes from mixtures
containing influenza B virus RNA (HA-1 and NA-1). In all
cases only a single product was detected by agarose gel elec-
trophoresis (data not shown). These cDNA probes correctly
hybridized to targets specific for each particular type and sub-
type of virus (e.g., Fig. 1, panels 11 and 12), and the hybrid-
TABLE 3. Primer pairs used to amplify influenza virus cDNAs
Fragment
Primer
Product
size (bp)
No. Sequence (5­3)
Location
(nta
)
A/HA1-1 1 CAGATGCAGACACAATATGTATAGG 74­587 513
2 GTTGTTTACATAGGACTTGCTCAG
A/HA1-2 3 ACAGAAATTTGCTATGGCTGAC 515­910 395
4 TCACATTCATCCATTGATGCATTTG
A/HA1-3 5 ATGGTATGCTTTCGCACTGA 834­1644 810
6 CGACAGTTGAATAGATCGCCA
A/HA2-1 7 GCAAAAGCAGGGGTTATACCA 1­555 554
8 AATTTGATTCTTTCTTTGTCAGCCA
A/HA2-2 9 TGTGTTACCCAGGCAGTTTC 353­874 521
10 CCTGAACTTCCTCTCTTCGATATT
A/HA2-3 11 AATATCGAAAAGAGGAAGCTCAGG 849­1340 491
12 CCACACATCTAGAAATCCATC
A/HA2-4 13 TGAATAGTGTGAAAAATGGAAC 1508­1662 154
14 CCAGTGACAGAGAACCTGCTAC
A/HA3-1 15 CATCATGCAGTGCCAAA 125­645 520
16 TTGGTCCGTGCTCGGGT
A/HA3-2 17 TTGGGGGGTTCACCACC 613­1057 444
18 GTTTCTCTGGTACATTCCGCA
A/HA3-3 19 GCAACAGGAATGCGGAA 1025­1732 707
20 CTCAAATGCAAATGTTGCACCTAA
A/NA1-1 21 ACTTCAGTGACATTAGCCGGCA 211­747 536
22 CGATCTTGAAAATTCTGTATGAGG
A/NA1-2 23 TGGCATGGGTTGGCTAA 528­1190 662
24 GAAACTCCCGCTGTATCC
A/NA1-3 25 TGGCAATAACTGATTGGTC 1151­1380 229
26 ACTTGTCAATGGTGAATGGCA
A/NA2-1 27 TTGCCATCCTGGCAACTACTGT 95­580 495
28 CAGCCATGCTTTTCCATCGTG
A/NA2-2 29 AACCCCTTATCGAACCCT 474­938 464
30 ATTTATATCTACGACCGGCCTATT
A/NA2-3 31 TGTTCCTGTTACCCTCGATATCC 850­1461 566
32 GATTGATGTCCGCTCCATCAG
A/MP-1 33 TGAAAGATGAGCCTTCTAACCGA 19­596 577
34 CTCCATAGCCTTAGCCGTAGT
A/MP-2 35 TGACAACAACCAACCCACTAA 521­923 402
36 TTTGGTACTCCTTCCGTAG
B/HA-1 37 GTGACTGGTGTGATACCACTAAC 153­665 512
38 CGGTATCAGAGTGGAACCCC
B/HA-2 39 AATGGCTTGGGCTGTCC 542­1018 476
40 TGCATGTTCTCCTGTGTAGTAAG
B/HA-3 41 GAGCAAGGTAATAAAAGGGTCC 908­1528 620
42 GAAGCATCCATTCCCTATGTCTAC
B/NA-1 43 CACTGTCATACTTATTGTATTCGGA 102­721 619
44 CGATGCAATTGCAGGCACTT
B/NA-2 45 CAATTGCATCGGGGGAG 709­1020 311
46 GCTTCCATCATCTGGTCTGG
B/NA-3 47 GGAAGCATAACAGGGCCTTG 1013­1352 339
48 GTTGCTGCTGAGTGCCAAGTC
B/MP-1 49 TTACACTGTTGGTTCGGTGG 105­630 525
50 GCCAGTTTTTGGACGTCTTC
B/MP-2 51 GGAAATACCTATAATGCTCGAACC 757­1084 327
52 CCTCCAAAACTGTTTCACCC
a
nt, nucleotides.
VOL. 39, 2001 DNA ARRAY DETECTION OF INFLUENZA VIRUSES 701
ization signals were again well above the background detected
at nonhomologous spots (Fig. 5).
DISCUSSION
Although DNA arrays have been most widely utilized by the
genomics research community to study gene expression pat-
terns and identify new genes, many authorities have speculated
that they might also prove useful in DNA-based diagnostics. In
this paper we have shown that robotically spotted DNA chips,
when used in conjunction with multiplex PCR methods, can be
used to facilitate typing and subtyping human influenza viruses.
Since the method exploits the specificity of both PCR and
DNA hybridization reactions, it offers a degree of accuracy
superior to those of many other methods commonly used to
detect PCR-amplified DNAs. Thus it should be attractive in
situations, such as medical diagnostics and forensics, where
further identification of a PCR-amplified DNA may be re-
quired. The method is obviously applicable to any pathogen
that can be cloned and arrayed and offers the advantage that
one can detect and differentiate DNAs contained in mixtures
of fluorescent PCR products (Fig. 1, panel 10). This suggests
that DNA arrays could streamline the detection of multiple
agents through parallel analysis of pools of PCR-amplified
DNAs.
Several features of gene chips, PCR, and fluorescent probes
facilitated the work outlined in this paper. First, PCR is well
known for its extraordinary sensitivity and can be used to
generate large quantities of probe DNA. Second, nucleotide-
linked Cy3 and Cy5 dyes are readily incorporated into PCR
products without greatly interfering with the efficiency of am-
plification or the yield of the probe. Third, these dyes are
intensely fluorescent and hybridization is easily detected at
levels well above background using modern confocally based
array readers. Finally, the hybridization and wash protocols
FIG. 4. Multiplex PCR amplification of influenza A and B virus
strains. The three multiplex primer sets described in Table 5 were
tested for the capacity to amplify a single, appropriately sized, DNA
product in reactions with the indicated cloned templates. For example,
primer mixture A (Table 5) produced a 510-bp product in a reaction
with an A/HA1-1 template (lane b), a 490-bp product in a reaction with
an A/HA2-3 template (lane e), primarily a 710-bp product in a reaction
with an A/HA3-3 template (lane h), and a 530-bp product in a reaction
with B/MP-1 template (lane k). PCR products were separated using a
1.2% agarose gel and stained with ethidium bromide.
FIG. 5. RT-PCR detection and differentiation of influenza virus
RNA templates. The multiplex primers indicated in Table 5 were
tested for the capacity to type and subtype the indicated influenza virus
strains. Probes were prepared in reaction mixtures containing crude
viral RNA, Cy3-dCTP, and an appropriate multiplex primer mixture
and hybridized overnight to microarrays. The signal intensity was cal-
culated as described in Materials and Methods (n  4). Signals marked
with an asterisk derive from perfectly matched probe-and-target pairs
(i.e., the probe is identical to the arrayed target), and the remaining
signals represent heterologous probe-and-target combinations (i.e.,
the target DNA came from a strain different from that tested in this
experiment).
TABLE 4. Fabrication of eight-by-eight influenza virus
cDNA arrays
Virus gene Row
DNA spotted at each column position for:
Influenza A virus Influenza B virus
1 and 2 3 and 4 5 and 6 7 and 8
HA 1 HA1-1 HA1-3 HA2-1 HA-1
2 HA2-2 HA2-3 HA2-4 HA-2
3 HA3-1 HA3-2 HA3-3 HA-3
NA 4 NA1-1 NA1-2 NA1-3 NA-1
5 NA2-1 NA2-2 NA2-3 NA-2
6 Buffer Buffer Buffer NA-3
MP 7 MP-1 MP-1 Buffer MP-1
8 MP-2 MP-2 Buffer E. coli
TABLE 5. Multiplex PCR primer combinations and
expected PCR products
Influenza
virus target
(virus type/
subtype)
PCR product generated by each primer mixture:
A (A/HA1-1, A/HA2-3,
A/HA3-3, B/MP-1)
B (A/NA1-1,
A/NA2-2, B/NA-1)
C (A/MP-1,
B/HA-1)
A/H1N1a
A/HA1-1b
A/NA1-1b
A/MP-1b
A/H1N2 A/HA1-1 A/NA2-2 A/MP-1
A/H2N1 A/HA2-3 A/NA1-1 A/MP-1
A/H2N2a
A/HA2-3b
A/NA2-2b
A/MP-1b
A/H3N1 A/HA3-3 A/NA1-1 A/MP-1
A/H3N2a
A/HA3-3b
A/NA2-2b
A/MP-1b
Ba
B/MP-1b
B/NA-1b
B/HA-1b
a
Virus typed and subtyped in this study.
b
See Fig. 4 for analysis of multiplex PCR products.
702 LI ET AL. J. CLIN. MICROBIOL.
differ little from the more-traditional methods used with other
nucleic acid binding membranes, and thus experience with
traditional methods is directly applicable to the glass arrays
used in these studies.
The preparation of DNA arrays used now fairly standard
technology. We fixed DNA to the slide surface using a 5-
amine-tagged oligonucleotide and carbodiimide cross-linker.
This method seems to provide a more stable array and works
well if, as was done here, cloned inserts can be reamplified
using a single pair of primers directed against flanking vector
sequences. However, the high cost of amine-linked primers
renders the method impractical if many different modified
primers are desired. In such situations we have employed non-
covalent binding methods with comparable success (J. Li and
D. H. Evans, unpublished data). To prepare DNA arrays ca-
pable of typing and subtyping human influenza A and B virus
strains, we designed 52 primers theoretically capable of ampli-
fying 26 different portions of the influenza A and B virus HA,
NA, and MP genes (Table 3). These primers amplified all but
two of the intended targets in standard RT-PCRs. One of the
two failures (B/MP-2) required primers that bind to sequences
poorly represented in the databases and thus may have been
badly designed; the failure of the other primer set (A/HA1-2)
is inexplicable. The 24 successfully amplified viral cDNAs nev-
ertheless provided a sufficiently redundant probe set for our
purposes and were cloned, sequenced, and arrayed.
These arrayed DNAs, each about 500 bp long, were readily
hybridized to probes prepared using the same cDNA templates
(Fig. 1 and 2), with signal-to-background ratios ranging from 6
to 30 (Fig. 2). No obvious hybridization was detected at non-
homologous targets, and no cross-hybridization was detected
among the different subtypes of the viruses. There was some
expected cross-hybridization seen when probes encoded se-
quences shared by two or more target sequences. Further anal-
ysis of this phenomenon showed that 100 bp of homology was
required to produce any detectable binding (Fig. 3). This effect
was almost certainly due to the stringent annealing conditions
we used to ensure maximal specificity, with the hybridization
temperature set 15°C higher than is recommended by the
manufacturer of the hybridization buffer (Roche) for use in
Southern blotting applications. This feature is useful, because
it ensures that primer-derived sequences common to both PCR
products and chip-bound targets will not cross-hybridize if
illegitimate sequences have accidentally misamplified. We also
noted that sensitivity was improved by increasing the size of the
target spot and by increasing the length of the fluorescent
probe (Fig. 1, panel 7). This last experiment also illustrates an
important advantage of arraying partially redundant gene frag-
ments rather than very large single cDNAs. The fact that a
1.6-kbp probe hybridized to three separate HA3 gene frag-
ments showed that the cDNA encoded sequences spanning the
entire gene. This provided an additional check on the identity
of the probe.
It was not obvious how sequence mismatches affected hy-
bridization efficiency. Using perfectly matched probes (i.e.,
probes derived from the same cDNA clones as target DNAs)
we noted signal-to-noise ratios varying from 6 to 30 over a
variety of gene fragments (Fig. 2). A similar range of signal-
to-noise ratios (5 to 24) was noted when heterologous probes
were prepared from viral isolates belonging only to the same
viral type and subtype as the target virus cDNAs (Fig. 5).
Analyzing the cause of this variation is difficult because it is
hard to measure the specific activities of fluorescent probes
and because there also appear to be target-specific variations
in hybridization efficiency even with identical probe-target
pairs (Fig. 2). However, it is encouraging to note that both
targets A/HA1-1 and A/NA-1 (derived from A/New Jersey/8/
76) hybridized to probes derived from virus strain A/Denver/
1/57 with signal-to-background ratios of 5.5 and 5.8, respec-
tively. Upon the conclusion of these experiments we directly
sequenced portions of these A/Denver/1/57-derived probes
and found that the sequenced portions had 86% sequence
identity with the target DNAs (GenBank accession no.
AF305216 and AF305218). Similarly, B/HA-1 and B/NA-1
probes (derived from strain B/Hong Kong/5/72) hybridized to
targets derived from strain B/Maryland/1/59 with signal-to-
background ratios of 19 and 24, respectively. In this case se-
quencing detected 96% sequence identity between probe
and target (GenBank accession no. AF305219 and AF305220),
suggesting that mismatches may reduce the amount of probe
bound under these stringent hybridization conditions. Never-
theless, it seems clear that, if PCR primers of sufficiently broad
utility can be designed, the great variation in influenza virus
gene sequences (Table 2) will not seriously interfere with the
application of this technology. We are currently expanding the
content of these arrays to permit detection of a much greater
selection of viral and other pathogens.
ACKNOWLEDGMENTS
We thank A. Hollis and B. Cooney for DNA sequencing.
This work was supported by an OMAFRA special research grant,
the Ontario Innovation Trust, and the Canadian Foundation for In-
novation. Research in D.E.'s laboratory was supported by NSERC and
CIHR grants.
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