J. Microbiol. Biotechnol. (2008), 18(6), 1164–1169
Development of Multiplex RT-PCR Assays for Rapid Detection and Subtyping
of Influenza Type A Viruses from Clinical Specimens
Chang, Hee Kyoung1, Jeung Hyun Park1, Min-Suk Song1, Taek-Kyu Oh1, Seok-Young Kim1,
Chul-Jung Kim2, Hyunggee Kim3, Moon-Hee Sung4, Heon-Seok Han1, Youn-Soo Hahn1, and Young-Ki Choi1*
1College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju 361-763, Korea
2College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Korea
3Division of Bioscience and Technology, College of Life and Environmental Science, Korea University, Seoul 136-701, Korea
4Department of Bio and Nano Chemistry, College of Natural Sciences, Kookmin University, Seoul 136-702, Korea
Received: November 12, 2007 / Accepted: January 22, 2008
We developed multiplex RT-PCR assays that can detect and
identify 12 hemagglutinin (H1-H12) and 9 neuraminidase
(N1-N9) subtypes that are commonly isolated from avian,
swine, and human influenza A viruses. RT-PCR products
with unique sizes characteristic of each subtype were
amplified by multiplex RT-PCRs, and sequence analysis
of each amplicon was demonstrated to be specific for each
subtype with 24 reference viruses. The specificity was
demonstrated further with DNA or cDNA templates from
7 viruses, 5 bacteria, and 50 influenza A virus–negative
specimens. Furthermore, the assays could detect and
subtype up to 105 dilution of each of the reference viruses
that had an original infectivity titer of 106 EID50/ml. Of
188 virus isolates, the multiplex RT-PCR results agreed
completely with individual RT-PCR subtyping results and
with results obtained from virus isolations. Furthermore,
the multiplex RT-PCR methods efficiently detected mixed
infections with at least two different subtypes of influenza
viruses in one host. Therefore, these methods could facilitate
rapid and accurate subtyping of influenza A viruses
directly from field specimens.
Keywords: Influenza A virus, multiplex RT-PCR, subtyping,
Avian influenza (AI) is a highly contagious disease caused
by type A influenza virus, which is an enveloped, single-
stranded, negative RNA virus of the Orthomyxoviridae
family. Influenza A virus frequently causes widespread
and fatal disease in birds as well as mammals, including
humans. Influenza A viruses can be classified into various
subtypes on the basis of antigenic differences between
the two surface glycoproteins, hemagglutinin (HA) and
neuraminidase (NA); there are 16 subtypes of HA (H1-16)
and 9 subtypes of NA (N1-9) . Amino acid sequence
identity among subtypes of HA and NA ranges from 25-
80% and 42-57%, respectively . All influenza A virus
subtypes have been found in aquatic and domesticated birds,
and a few subtypes have been recovered from mammals.
Influenza A viruses that have infected humans during the
past 90 years have been limited to the H1, H2, and H3
subtypes. However, human infections with several AI
subtypes such as H5N1 [4, 15, 21, 22], H7N7 , and
H9N2 [3, 8, 16] have occurred, thus demonstrating direct
crossing of the species barrier. Therefore, there is a great
need for more rapid and precise methods to detect HA and
NA subtypes irrespective of the species (avian, swine, and
Methods for detecting and subtyping influenza viruses
utilize the propagation of virus in tissue culture or embryonated
eggs before subtyping by hemagglutination inhibition (HI)
 and neuraminidase inhibition (NI) tests, which use a
monospecific antiserum to each subtype . Although
virus propagation in tissue culture or embryonated eggs is
sensitive and accurate, it requires several days for a viable
virus to cause observable cytopathic effects; thus, such
assays are time-consuming and laborious. Other diagnostic
tests have also been used, such as immunofluorescence
staining and enzyme-linked immunosorbent assay (ELISA)
based on the detection of nucleoprotein antigen .
ELISAs can rapidly detect viruses, but the sensitivity is
comparatively poor. Molecular techniques such as PCR-
based methods, however, have enabled major advances in
the speed and sensitivity of the laboratory diagnosis of
viral infections. Specifically, these methods have higher
sensitivity (93%) for influenza A viruses than cell culture
methods (80%) and ELISA (62%) . Among the 16 HA
subtypes, only H5 and H7 are highly virulent in poultry,
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RAPID DETECTION AND SUBTYPING OF INFLUENZA VIRUSES
and the rest of the viruses cause a much milder, primary
respiratory disease known as low-pathogenic avian influenza
. However, no influenza virus subtype can be ruled out
as a candidate for a potential pandemic, because all three
pandemic influenza viruses in the 20th century originated
directly or indirectly from avian influenza viruses.
We have monitored influenza viruses from domesticated
and migratory birds in Korea since 2004. Of the 144
subtypes, the H1 to H12 subtypes of the HA gene and N1
to N9 subtypes of the NA gene have been commonly
detected from migratory birds and domesticated animals in
Korea (unpublished data). To facilitate more rapid detection
of these subtypes, we have developed multiplex RT-PCR
assays that use appropriate primer mixtures specifically
designed to detect and identify 12 HA (H1-H12) and 9 NA
(N1-N9) subtypes of avian, swine, and human influenza
viruses. These multiplex RT-PCR assays were used to
investigate the prevalence of each avian influenza virus
subtype and to rapidly detect highly pathogenic avian
influenza (HPAI) from clinical specimens and viral isolates.
MATERIALS AND METHODS
Isolation of Viruses
Influenza virus strains were isolated in embryonated eggs and
MDCK cells from tracheal swabs and fecal samples of animals
during 2004-2007. Tracheal swabs and fecal samples were collected
into tissue culture medium containing penicillin G (2×106U/l),
polymyxin B (2×106U/l), gentamicin (250 mg/l), nystatin (0.5×
106U/l), ofloxacin HCl (60 mg/l), and sulfamethoxazole (0.2 g/l).
Specimens were inoculated into MDCK and embryonated eggs and
influenza viruses purified as previously described [10,14]. Virus isolates
were subtyped with a panel of reference antisera recommended by
the World Health Organization (http://www.who.int/csr/resources/
publications/en/#influenza). One hundred eighty-eight type A influenza
virus isolates, cultured from cells or embryonated eggs, 85 virus-
positive fecal specimens from birds, and 40 lung samples from
swine were selected from archived isolates/samples at Chungbuk
National University. Thirty-five influenza virus-negative samples (20
from fecal specimens of birds, and 15 swine lung tissues) were also
obtained. These samples were collected from swine and domestic
poultry between 2004 and 2007. Approximately 1g of fecal specimens
was placed in a tube with 3-5 ml of Eagle’s minimum essential
medium (MEM) and vortexed vigorously. Approximately 10%
suspension of each lung homogenate was also prepared in Eagle’s
MEM. The suspensions of fecal specimens or lung homogenates
were centrifuged at 800×g for 20min, and 200µl of the supernatants
was used for virus isolation and RNA extraction. Virus-positive
allantoic or cell culture fluids were harvested and stored as stocks at
-80oC until used.
Extraction of RNA and Synthesis of cDNA
Viral RNA was extracted using the RNeasy Mini kit (Qiagen,
Valencia, CA, U.S.A.) according to the manufacturer’s instructions.
Briefly, 200 µl of the specimens was mixed with 550 µl of RLT
buffer and incubated for at least 5 min at room temperature. After
the addition of 550 µl of absolute ethanol, the mixture was vortexed
and applied to a spin-column. After the washing and drying steps,
RNA was eluted in 40 µl of RNase-free water. Reverse transcription
was carried out under standard conditions by using influenza-
specific primers .
Multiplex PCR Reaction
The PCR reaction in a 50 µl volume contained 5 U of TaKaRa EX
Taq (Takara Bio Inc., Shiga, Japan), 6 µl of 20 mM Mg2+, and 3 µl
of 2.5 mM of each dNTP, appropriate concentrations of template
cDNA, and 1 µl of 10 pM primer mixture (Table 2). Each PCR
product was amplified by the following conditions: denaturation step
for 5 min at 94oC, 30 cycles of denaturation at 94oC for 30 s,
annealing at 58oC for 30 s, and extension at 72oC for 60 s, followed
by a final extension step at 72oC for 10 min. The amplified products
were analyzed by 0.8% agarose gel electrophoresis.
The multiplex RT-PCR assays were also tested for specificity
using five bacteria (104CFU of each bacterium) and seven viruses
(104PFU of each virus) that commonly infect swine. Bacterial
pathogens tested were Mycoplasma hyopneumoniae (ATCC 27719)
and M. hyorhinis (ATCC 23839), and field isolates of Streptococcus
suis, Hemophilus parasuis, and Bordetella bronchiseptica. Viruses
tested were porcine reproductive and respiratory syndrome virus
(ATCC VR-2332), transmissible gastroenteritis virus (ATCC VR-743),
encephalomyocarditis virus (EMCV-CBNU) , porcine parvovirus
(NADC-8), pseudorabies virus (Shope strain), avian pneumovirus
(CNV-PL1), and avian New Castle disease viruses (clone 4 vaccine
strain and several field isolates).
The DNA fragments were extracted and purified with a QIAquick
gel extraction kit, and sequencing of the amplified DNA was
performed at Macrogen (Seoul, S. Korea) with an ABI 373 XL
DNA sequencer (Applied Biosystems, Foster City, CA, U.S.A.).
DNA sequences were compiled and edited using the Lasergene
sequence analysis software package (DNASTAR, Madison, WI,
Design of HA and NA Primers
At least 20 sequences encompassing human, avian, and
swine HA and NA sequences of each subtype were
selected from the Flu Database (http://www.flu.lanl.gov/)
and aligned to determine common regions. According to
the similarity and length of the nucleotide sequences, we
designed primer sets for 12 HAs (H1-H12) and 9 NAs
(N1-N9) (Table 1). Initially, we attempted to amplify 12
HA and 9 NA genes using all 12 HA and 9 NA primer sets
in one tube with known reference viruses. However, the
amplification was relatively poor compared with the
efficiencies attained using individual primer sets. Moreover,
because of nonspecific bands and overlapping DNA fragments,
precise subtyping was not possible. Therefore, we optimized
the number of primers per mixture based on the criteria of
amplification efficiency, specificity, and the ability to
1166Chang et al.
distinguish products by size. Five to six primers were
selected to comprise three HA and NA primer mixtures.
The three groups (I, II, and III) of HA and NA genes
containing 5-6 primers demonstrated the best conditions
for sensitivity and specificity for subtyping of HA and NA
genes (Table 2). Each multiplex HA primer could detect
the following subtypes of HA genes: group I (H3, H5, H7,
and H9), group II (H1, H2, H4, and H8), and group III (H6,
H10, H11, and H12). Similarly, each multiplex NA primer
could amplify the following subtypes of NA genes: group I
(N3, N4, and N6), group II (N5, N8, and N9), and group
III (N1, N2, and N7) (Table 2).
Detection and Subtyping of Influenza A Virus
To evaluate our multiplex RT-PCR methods, we tested our
multiplex RT-PCR with 24 reference influenza type A
viruses isolated from swine, and wild and domesticated poultry
in Korea (Table 3). Twelve HA and nine NA subtypes of
the influenza viruses could be clearly differentiated by the
size of the amplified DNAs (Fig. 1). Qualitative comparison
between the multiplex amplified bands and individually
amplified bands shown in Fig. 2 demonstrated that the
multiplex primers were capable of amplifying the appropriate
PCR amplicons with the same efficiency.
The specificity of RT-PCR products was confirmed by
sequencing (data not shown). The specificity was demonstrated
further by testing with DNA or cDNA templates from seven
viruses and five bacteria (listed in Materials and Methods)
or mock-infected MDCK cells. None of the multiplex RT-
PCR reactions was positive when the primers were tested
with non-influenza A virus specimens. To evaluate the
sensitivity of the multiplex RT-PCR, the primer sets were
tested with 10-fold serially diluted RNAs extracted from
200 µl of each virus stock that was adjusted to contain
106 EID50/ml of 24 reference influenza A viruses.
Amplification could be visualized with a 105 dilution of
each of the reference viruses (data not shown).
Evaluation of the Multiplex RT-PCR Assay with Field
To evaluate the multiplex RT-PCR using field specimens, we
tested 188 influenza A virus isolates, 40 lung homogenates,
and 85 fecal or tracheal specimens from swine, and wild
or domesticated poultry in Korea (Table 4). The results
for detection and subtyping of influenza A viruses from
field cases using multiplex RT-PCR were in complete
agreement with those for virus isolation in MDCK cells or
chicken embryonated eggs and with amplification via RT-
PCR with individual primers. Six virus isolates and 11
fecal/tracheal swabs were diagnosed as a mixture of at
least two different subtypes of influenza viruses (Fig. 2). In
addition, no positive amplification was observed when the
Table 1. The multiplex PCR primers used to amplify avian influenza viruses.
NameSequence of oligonucleotideNameSequence of oligonucleotide
H10 1342R 5'-TCA ATT GTG TGC TGA TTT TCC ATT-3'
H11 794R5'-GCA CCA TTW GAC TCA AAT GTT ATT-3'
H12 429R5'-CAG TGT ATG TGA CAT TCC ATT TGG-3'
5'-AGC AAA AGC AGG GGA-3'
5'-AGC AAA AGC AGG GGT-3'
5'-TAC CAY CCA TCT ATC ATK CCT G-3'
5'-TCA AGT GTC CTC TCA TTT TCC AT-3'
5'-RTA GRG CAT CTA TYA RTG TRC A-3'
5'-GYA CAT CAA ATG GRT ARC AAG T-3'
5'-ARC TYC TCT TTA TTG TTG GGT A-3'
5'-AAT TCA TRC TTT CAG TTC CCA T-3'
5'-GCW GCA GTT CCY TCY CCT TGT-3'
5'-TTT TGA ATT ATT CTG CCA TGG CT-3'
5'-GRG CRA TTA RAT TCC CAT TGG A-3'
N6 1219R 5'-CCT GAG TAY CCY GAC CAG TTT TG-3'
N7 1378R 5'-CCG GAC CCA ACT GGG AMT GGG C-3'
N8 1342R 5'-CTC CAC ACA TCA CAA TGG AGC T-3'
N9 451R5'-TCG TGT ATT GTT CCA TTT GAG TGT-3'
5'-AGC AAA AGC AGG AGT-3'
5'-AGC AAA AGC AGG TGC-3'
5'-AGC AAA AGC AGG GTG AAA ATG-3'
5'-AGC AAA AGC AGG GTG ATT GAG AAT-3'
5'-AGC AAA AGC AGG GTC-3'
5'-GCA CTT GCY GAC CAA GCA ACW GA-3'
5'-GCR CTY CCA TCA GTC ATT ACT ACT-3'
5'-ATT ATT GTT GTT GTT ATG TTG TT-3'
5'-TCT GTT ATT ATA CCA TTR TAT TT-3'
5'-GGA CCA TCY GTC ATT ACC CAA TAA-3'
*A+G: RC+T: Y A+C: MG+T: KG+C: S A+T: W A+T+C: HG+A+T: DG+T+C: BG+A+C: V A+G+C+T: N
Table 2. Components of HA and NA primer mixtures of groups I, II, and III.
HA primer mixturesNA primer mixtures
III IIII IIIII
H3 (267 bp)0,
H5 (545 bp)0,
H7 (1,155 bp)
H9 (796 bp)0,
H1 (1,077 bp)
H2 (1,325 bp)
H4 (342 bp)0,
H8 (848 bp)0,
H6 (669 bp),00
H10 (1,342 bp)
H11 (794 bp)0,
H12 (429 bp)0,
N3 (233 bp)0,
N4 (644 bp)0,
N6 (1,219 bp)
N5 (726 bp)0,
N8 (1,342 bp)
N9 (451 bp)0,
N1 (546 bp)0,
N2 (734 bp)0,
N7 (1,378 bp)
RAPID DETECTION AND SUBTYPING OF INFLUENZA VIRUSES
multiplex RT-PCR assays were performed on 50 influenza
A virus-negative specimens (30 fecal/tracheal swabs and
20 lung tissues) (Table 4).
To further confirm whether the PCR products amplified
using multiplex primers were the actual HA or NA subtype
of the influenza virus, we sequenced the purified genomic
DNA. All amplified DNAs had the correct sequence for
the corresponding subtype (data not shown).
To date, influenza A viruses comprise 16 HA and 9 NA
subtypes, which implies a total of 144 possible combined
subtypes. Currently, human infections of highly pathogenic
avian H5N1 and H7N7 subtypes are being reported, and
certain avian influenza A viruses seem to cross the species
barrier to infect other species that originally were not
known as the natural host . Given this scenario, we
need a simple, rapid, and accurate method to detect the
various subtypes of influenza A viruses regardless of
species origin. Many multiplex PCR methods have been
developed and applied to various viral agents to satisfy the
need for rapid and economical detection and diagnosis of
viral infections. However, most of the methods have focused
only on highly pathogenic or commonly found subtypes,
such as H1, H3, H5, H7, and H9 influenza viruses [5, 12,
17, 24]. Furthermore, we acknowledge the fact that no
influenza virus subtype can be ruled out as a candidate for
a potential next pandemic outbreak. Therefore, we devised
a simple and rapid multiplex RT-PCR method to identify
12 HA subtypes (H1-H12) and 9 NA subtypes (N1-N9),
which were commonly isolated in South Korea. After
various attempts to select the appropriate number of
primers to be used in one mixture, four HA subtypes and
Table 3. The reference influenza A viruses tested for multiplex
No.IsolatesHA typeNA typeHost
Fig. 1. Detection and subtyping of type A influenza virus by
multiplex RT-PCR assay.
The virus strains representing the 12 hemagglutinin (HA) and 9
neuraminidase (NA) subtypes used for RT-PCR are shown in Table 3. A.
PCR reactions for detection and subtyping of H1 to H12 subtypes using a
specified primer mixture; group I (H3, H5, H8, and H9), group II (H1,
H2H4, and, H8), and group III (H6, H10, H11, and H12). B. PCR reactions
for detection and subtyping of N1 to N9 subtyping using a designated
primer mixture; group I (N3, N4, and N6), group II (N5, N8, and N9), and
group III (N1, N2, and H7). The M1, M2, and M3 lanes represent the PCR
reaction with all groups I, II, and III virus mixtures in one tube, respectively.
Fig. 2. Detection and subtyping of dual infections by multiplex
RT-PCR (A) and single PCR (B) assays of avian influenza A
viruses from fecal specimens of wild ducks.
A. PCR reaction with HA group I primer mixture (lanes 1-2) and NA
group III primer mixture (lanes 3-4). Lane M, Hi-Low size DNA marker;
lane 1, dual positive of H3 and H9; lane 2, dual positive of H5 and H7; lane
3, dual positive of N1 and N2; lane 4, dual positive of N1 and N7. B.
Single PCR assay using individual primer set of each HA and NA subtype
with the same cDNA template used in multiplex RT-PCR assay. The result
of the single PCR reaction with individual primer set completely agreed
with the multiplex RT-PCR assay.
1168 Chang et al.
three NA subtypes were chosen as the optimal number of
subtypes to be detected in one tube. Comparison of the
visual images of amplification utilizing individual primer
sets and multiplex primer sets revealed that multiplex PCR
methods were as specific and efficient as individual PCR
methods (Fig. 1). In addition, our method has the advantages
of being able to conserve time and reagents, considering
that 3 to 4 subtypes could be distinguishably identified in a
single amplification run for each of the NA and the HA
genes, respectively. The cost-effective feature, however,
does not necessarily affect or compromise the proven
specificity and sensitivity of the procedure. The multiplex
RT-PCR assays developed in the present study could
differentiate 12 HA and all 9 NA subtypes of influenza A
viruses from cultured virus isolates and, more importantly,
field specimens (Table 4). As such, these assays may facilitate
influenza virus diagnosis with easier identification and
more rapid subtyping compared with other methods. It is
important to note that multiple infections have been detected
in virus isolates and clinical field samples, indicating the
possibility of concurrent or sequential infection in host
animals and the occurrence of more complex influenza
virus subtypes or genotypes (Fig. 2). Given the multiple
and complex infections caused by many of the known
influenza viruses, the application of our multiplex PCR
assays will also contribute to the rapid detection and
diagnosis of recent reassortment events among avian, swine,
and human viruses, and hopefully prevent the further
spread of these viruses.
This work was supported in part by an internal fund from
the Chungbuk National University in 2006 grant No.
R01-2005-000-10585-0 from Basic Research program of
the KOSEF, and by Green Cross. Corp. We would like
to express our appreciation to Young-Chul Jeung for
providing excellent field support and Philippe Noriel Q.
Pascua for editorial assistance.
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Table 4. Detection and subtyping of type A influenza virus isolates, virus-positive/negative fecal/tracheal swabs or lung samples from
poultry and pigs by multiplex RT-PCR assays.
No. of positive
with individual PCR (%)
No. of multiple
Virus-positive by virus isolation or HA tests
Virus-negative by virus isolation or HA tests