A Multiplex Reverse Transcriptase-PCR Assay for the Genotyping of Avian Infectious

Bronchitis Viruses
Author(s): Hui-Wen Chen and Ching-Ho Wang
Source: Avian Diseases, Vol. 54, No. 1 (March 2010), pp. 104-108
Published by: American Association of Avian Pathologists
Stable URL: http://www.jstor.org/stable/40600004 .
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AVIAN DISEASES 54:104-108, 2010
A
Multiplex
Reverse
Transcriptase-PCR Assay
for the
Genotyping
of Avian Infectious
Bronchitis Viruses
Hui-Wen ChenA and
Ching-Ho WangAB
ASchool of
Veterinary
Medicine, National Taiwan
University,
No. 1, Sec. 4, Roosevelt Rd.,
Taipei
10617, Taiwan
Received 9
June 2009;
Accepted
and
published
ahead of
print
27 October 2009
SUMMARY. Infectious bronchitis viruses
(IBVs)
in Taiwan have been divided into two
genogroups,
Taiwan
group
I
(TW-I)
and Taiwan
group
II
(TW-II).
Heterologous Mass-type
strains are
widely
used as vaccines in the field. This work
reports
on a
rapid
and reliable
multiplex
reverse
transcriptase-polymerase
chain reaction
(mRT-PCR) assay
for the
genotyping
of IBVs.
Multiplex
primer
sets were
designed
to
amplify
the
region covering hypervariable regions
1 and 2 of the S 1
glycoprotein gene.
Several local
strains and
commercially
available vaccines were used for
evaluating
the viral
genotyping assay,
and a number of field isolates were
examined for clinical
application.
The results showed that all of the examined IBVs were
accurately genotyped by identifying
the
corresponding
bands on
agarose gels (TW-I: 322
bp,
TW-II: 161
bp,
Mass
type:
256
bp)
after the mRT-PCR,
in
agreement
with
the viral
genome sequence
data. The mRT-PCR
assay
was able to detect viral RNA
copies
as low as 103, 105, and 103 for the TW-I,
TW-II, and Mass-
type
strains,
respectively.
The mRT-PCR
assay accurately
detected and discriminated vaccine viruses from wild-
type
strains in the field. This
assay may
be beneficial for virus identification and differentiation in routine disease surveillance.
RESUMEN.
Ensayo multiple
de
transcripcin
reversa
y
reaccin en cadena de la
polimerasa para
la
genotipificacin
del virus de
la
bronquitis
infecciosa de las aves.
Los virus de la
bronquitis
infecciosa
(IBV)
en Taiwan se han dividido en dos
genogrupos,
el
grupo
Taiwan
(TW-I) y
el
grupo
Taiwan II
(TW-II). Cepas heterlogas
del
tipo
Massachusetts son
ampliamente
utilizadas como vacunas en el
campo.
En este
trabajo
se describe un
procedimiento multiple
de
transcripcin
reversa
y
reaccin en cadena de la
polimerasa (MRT-PCR) rpido y
confiable
para
la
genotipificacin
de
cepas
del virus de la
bronquitis.
Se disefiaron los iniciadores del mtodo
multiple para
amplificar
la
region que
abarca las
regiones hipervariables
1
y
2 del
gen
de la
glicoproteina
SI. Se utilizaron varias
cepas
locales
y
vacunas
disponibles
comercialmente
para
evaluar el
ensayo
en la determination del
genotipo
viral
y
vrios aislamientos de
campo
fueron analizados
para
evaluar la
aplicacin
clnica del mtodo. Los resultados mostraron
que
todos las
cepas
del virus de
bronquitis
examinadas fueron
tipificadas geneticamente
de manera
precisa
mediante Ia identificacin de Ias bandas
correspondientes
en
geles
de
agarosa
(una
banda de 322
pb para
el
grupo
TW-I, banda de 161
pb para
el
grupo
TW-II
y
una banda de 256
pb para
el
tipo
Massachusetts),
el
procedimiento multiple
MRT-PCR mostro concordncia con el anlisis de Ias secuencias dei
genoma
viral. El
procedimiento
MRT-PCR fue
capaz
de detectar
copias
de ARN viral en concentraciones tan
bajas
cortio IO3, IO5
y
IO3
para
el TW-
I, TW-II
y
el
tipo
Massachusetts,
respectivamente.
El mtodo MRT-PCR detecto
y
discrimino con
precision
virus vacunales de
cepas
silvestres en el
campo.
Este
ensayo puede
ser beneficioso
para
Ia identificacin del virus
y
la diferenciacin en
programas
rutinarios de
vigilncia
de enfermedades.
Key
words:
genotyping,
infectious bronchitis virus,
multiplex
RT-PCR, Taiwan
Abbreviations: AIV
=
avian influenza virus; DEPC
=
diethyl pyrocarbonate;
DIVA
=
differentiating
infected from vaccinated
animals; HVR
=
hypervariable region;
IBV
=
infectious bronchitis virus; M
=
membrane
protein;
mRT-PCR
=
multiplex
reverse
transcriptase-polymerase
chain reaction;
=
nudeocapsid protein;
NDV
=
Newcasde disease virus; S
=
spike protein;
TW-I
=
Taiwan
group
I; TW-II
=
Taiwan
group
II
Avian infectious bronchitis virus
(IBV)
belongs
to the
family
Coronavindae,
group
3 Coronavirus and is one of the most
important
pathogens
in
poultry.
IBVs can
replicate
in tissues of the
respiratory,
intestinal, and
urogenital
tracts. Infected chickens
may
show reduced
feed
efficiency
and
poor egg production (8,9).
This disease was first
observed in the United States in 1 930
(29)
and has caused tremendous
economic losses to the
poultry industry
worldwide
(7,11).
IBV contains a
positive
sense,
single-stranded
RNA
genome
that is
approximately
27.6 kb
long.
As much as 20 kb is a
major portion
of the
RNA-dependent
RNA
polymerase gene,
and the
remaining gene
region, approximately
7 kb,
expresses
three structural
proteins
and
several smaller nonstructural
proteins (24).
The
spike protein (S),
one
of the structural
proteins,
is
heavily glycosylated
and
posttranslationally
cleaved into two subunit
proteins,
SI and S2. The
peplomer
forms the
most exterior
part
of the virion and functions as the
binding receptor
for host cells
(24).
The SI
protein
is
responsible
for viral
infectivity
and
Corresponding
author. E-mail:
chingho@ntu.edu.tw
inducing neutralizing
and
hemagglutination-inhibition
antibodies
(9,22).
The S2 subunit constitutes the stalk of the viral
spike
and
also carries one
virus-neutralizing epitope (21).
Coronaviruses
possess
diverse host
ranges
or cell
tropisms through
variations in the S
gene
(8);
the S 1
gene sequences
are
highly
variable, and mutations in the S 1
gene
may
result in altered
antigenicity
(24).
Few differences in SI amino
acid residues
(2%
to
3%)
can cause a
change
in
serotype (10).
Two
other structural
proteins,
the membrane
glycoprotein (M),
which is
associated with virus
assembling
and
budding,
and the
nudeocapsid
protein (N)
interacts with the viral
genome
to form the
nudeocapsid.
The
gene
is located at the extreme 3' end of the
genome
and is
relatively
conserved
(24).
Like most RNA viruses, IBVs evolve and have
significant genetic
diversity (24).
Dozens of viral sero
types
and
genotypes
have been
reported (8).
Although
vaccines have been used to control IB,
outbreaks still occur
(11).
Several viral
genotypes
can exist in a
region {AyG),
and the co-circulation of viruses
may
contribute to
more viral variants and result in new outbreaks. Therefore, viral
epidemiology
is
frequently
examined
(1,15,28,35).
104
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Multiplex
RT-PCR for IBV
genotyping
105
Table 1. Local IBVs used for mRT-PCR evaluation.
Strain Year isolated
GenotypeA
Accession no. Reference
1171/92 1992 TW-I
DQ646406 (17)
2575/98 1998 TW-I
DQ646405 (17)
3071/03 2003 TW-I EU822339 (17)
3468/07 2007 TW-I EU822336 (12)
2296/95 1995 TW-II
DQ646404 (17)
3263/04 2004 TW- EU822338 (12)
AGenotype
was determined based on the Si
gene.
TW-I
=
Taiwan
group
I; TW-II
=
Taiwan
group
II.
A
strong diagnostic
tool is a must for
conducting large-scale
viral
surveillance. Viral strain
typing
is crucial for
implementing
control
measures and
understanding
the
epidemiology
of local IBVs. Reverse
transcriptase-polymerase
chain reaction
(RT-PCR)
has been
extensively
used in IBV identification
(14).
For
group-specific
diagnosis,
more conserved IBV
gene regions
such as the M or
gene
can be
amplification targets
(3,16,36).
In
contrast, the SI
gene
has
three
hypervariable regions
(HVRs) (19,25).
The SI
gene-based
RT-
PCR can be used as a
genotype-specific assay (1,5,20,23,35).
However,
regardless
of which
gene
is
targeted,
further
techniques
such as DNA
sequencing,
restriction
enzyme length polymorphism,
or
hybridization
are
mostly
needed to obtain
strain-typing
results.
These
manipulations
are
usually
laborious and
costly.
IBVs have been
reported
in Taiwan since 1956, and
they
are still
the main infectious
agents
in chicken farms. The viral
genotypes
in
Taiwan have been divided into two
groups,
Taiwan
group
I
(TW-I)
and Taiwan
group
II
(TW-II),
both of which exist as exclusive virus
populations
in the world
(17,32).
Since a
heterologous
Mass-
serotype
vaccine has been used in Taiwan for a decade,
group-
specific
virus identification is sometimes
confusing.
We
report
on a
multiplex
RT-PCR
(mRT-PCR) assay
that can be used as a direct
and
rapid
viral
genotyping technique.
Several local strains and
commercially
available vaccines were
employed
for
evaluating
the
viral
genotyping assay.
The limit of detection was determined
using
the known
copy
number of RNA. For clinical
application,
1 3 field
isolates were also introduced to examine the
accuracy
of the
assay.
MATERIALS AND METHODS
Viruses and vaccine strains. All of the viruses used in this
study
were
confirmed to be IBVs
by
RT-PCR with
gene-specific primers (17)
that
detect IBVs
universally.
These IBVs were
propagated
in the allantoic
cavity
of 9- to 11
-day-old specific pathogen
free
embryonated eggs
(AHRI,
Council of
Agriculture,
Tamsui, Taiwan).
Each
egg
received a 0.2 ml
inoculation of the seed virus or the tissue
homogenates.
Infected allantoic
fluid was harvested 48-72 hr
post-inoculation
and stored at
-
80 C until
use. The local IBVs used in this
study
are listed in Table 1. IBV vaccine
strains 120
(Abie,
Beit Shemesh, Israel), Ma5 (Intervet, Boxmeer, the
Netherlands),
M4l
(Merial, Taipai, Taiwan), 4/91 (Intervet),
and Conn
(a
kind
gift
from Dr. Lu, AHRI)
were included in this
study.
Viral RNA extraction. Viral RNA was extracted from 140

of
virus-containing samples using
the
QIAamp
Viral RNA Mini kit
(Qiagen,
Valencia, CA).
The
resulting
RNA was dissolved
using
a
volume of 60

of
diethyl pyrocarbonate (DEPC)
-treated water.
Primer
design.
The nucleic acid
sequence alignments
of the TW-I,
TW-II and
Mass-type
viruses were
generated using
the DNAStar
software
(DNAStar, Madison, WI).
The
multiplex primer
sets were
designed
to
target
the SI
gene region
(Table 2).
A fixed forward
primer,
rC2U
(17,18),
was used, and three reverse
primers
were
designed
from
the
regions
conserved
among only
one
genogroup
that were diverse from
the other two. A BLAST
analysis
of the GenBank database was
performed
to assure the
specificity
of the
primers.
IBV
genotyping multiplex
RT-PCR. The
genotyping multiplex
RT-
PCR was
performed
in one
step
in a
thermocycler GeneAmp
PCR
system
2700
(Applied Biosystems,
Foster
City, CA).
A total volume of
25
of reaction mixture was
prepared, including
5

of viral RNA
template,
0.5
Phire Hot Start DNA
polymerase (Finnzymes Oy,
Espoo, Finland), 5
of 5X Phire reaction buffer
(Finnzymes),
600

dNTPs
(GeneTeks
BioScience Inc.,
Taipei, Taiwan), 0.5
upstream primer (Tri-I
Biotech Inc.,
Taipei, Taiwan), 0.5
three
downstream
primers (Tri-I
Biotech
Inc.),
8 U RNaseOut
M
recombi-
nant ribonuclease inhibitor
(Invitrogen,
Carlsbad, CA),
20 U M-MLV
reverse
transcriptase (Invitrogen),
and 7.2

of DEPC-treated water.
Reverse
transcription
was carried out at 40 C for 30 min and followed
by
the activation of DNA
polymerase
at 98 C for 30 sec. PCR was then
performed
for 35
cycles
of 98 C
(5 sec), 58.4 C
(5 sec),
and 72 C
(10 sec).
The reaction was
completed
with the final
polymerization step
at 72 C for 1 min. The obtained DNA
products
were
analyzed using
electrophoresis
in 2%
Agarose
I
gel
(Amresco Inc., Solon, OH)
stained
with ethidium bromide.
Full SI
gene cloning
and RNA in vitro
transcription.
The full SI
genes
of IBV strain 2575/98 (TW-I), 2296/95 (TW-II),
and H120
(Mass type)
were
amplified using
RT-PCR as
previously
described
(17).
Following
the instructions of the
yT&A Cloning
Vector Kit
(Yeastern
Biotech,
Taipei, Taiwan),
the recombinant
plasmid
DNA
containing
the
corresponding
S 1
gene
was constructed, transformed into ECOS 9-5
competent
cells
(Yeastern Biotech),
and screened
by colony
PCR. The
insert nucleotide
sequence
was confirmed
by
DNA
sequencing
(Tri-I
Biotech
Inc.).
The
plasmid
was then
purified using
the
QIAprep Spin
Miniprep
kit
(Qiagen)
and linearized
using
BarnHl
(Takara
Bio Inc.,
Shiga, Japan) digestion.
An amount of 2
g
linear
plasmid
DNA served
as the
template
for in vitro
transcription,
which was
performed using
the
T7 RNA
transcription
kit
(Fermentas, Hanover, MD) according
to the
manufacturer's instructions. Afterwards, the DNA
template
was
removed
using
the Ambion TURBO
ONK-free
kit
(Applied
Biosystems)
at 37 C for 2 hr. PCR without the RT
step
was
performed
to assure
complete
DNA
template
removal. The
resulting
RNA was
quantified using Spectrometry (GE Healthcare, Oslo,
Norway),
and the
copy
number of RNA
transcripts
was calculated as follows:
(6
23
total
weight [g]
of
RNA)/(343
X base number of
RNA).
The RNA
aliquots
were stored at -80 C.
Partial SI
gene sequencing
and
sequence analyses.
The 5' end of
the SI
gene
was
amplified
and
directly sequenced
from RT-PCR
products (17). Sequence analyses
and
phylogenetic analyses
were
performed
as
previously
described
(12).
Detection limit and
accuracy.
The detection limit of the mRT-PCR
was evaluated with in vitro transcribed RNA. Ten-fold serial dilutions of
Table 2. Primer sets used in mRT-PCR.
Primer
Sequence (5'
-
*
3')
Position
Targeted type
Sense
Amplicon
rC2U TGGTTGGCAYTTACAHGG 114-131A TW-I, TW-II, Mass +
TWI-R2 GAATAAAGTATGATTCCGCAT 415-435B TW-I
-
322
bp
TWII-R2 AAGTCATACCATTATTCGGAG 254-274c TW-II
-
161
bp
H120-R2 AGTTATAGGACACCCAACAT 350-369A Mass
-
256
bp
ANucleotide
position
in the S
gene
of the strain H 120
(GenBank
accession no.
EU822341).
BNudeotide
position
in the S
gene
of the strain 2575/98 (GenBank
accession no.
DQ646405).
cNudeotide
position
in the S
gene
of the strain 2296/95 (GenBank
accession no.
DQ646404).
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106 H.-W. Chen and C.-H.
Wang
Table 3. Field isolates used for mRT-PCR clinical
application.
Field isolate Year isolated
GenotypeA
Chicken
embryo passages
3339/05 2005 TW-I 4
3368/05 2005 TW-I 2
3369/05 2005 TW-I 2
3370/05 2005 TW-I 3
3371/05 2005 TW-I 3
3372/05 2005 TW-I 3
3373/05 2005 TW-I 3
3374/05 2005 China-like 3
3376/06 2006 TW-I 3
3381/06 2006
JMK-like
3
3382/06 2006 TW-I 3
3384/06 2006 TW-I 3
3385/06 2006 TW-I 3
Genotype
was determined based on the SI
gene.
TW-I
=
Taiwan
group
I.
RNA
transcripts containing
102 to 106
copies per
5
were used as
templates
in the mRT-PCR. Three
types
of RNA
transcripts
were first
examined
individually
to
investigate
the limit of detection. This
step
was
followed
by
the use of a
paired
combination of two
types
of RNA
transcripts
at the ratios of 1:1 and 1:10
(equal
and 10-fold the number
of
copies
defined as the detection limit,
respectively)
as the
templates
in
the mRT-PCR. The detection
accuracy
was examined
using
other avian
respiratory agents.
Viral RNAs of one avian influenza virus
(AIV) strain,
A/chicken/Taiwan/2838V/00 (33),
and a Newcastle disease virus
(NDV) strain, TW-2/00
(13),
were extracted as described above and
assayed using
mRT-PCR.
Clinical
application.
Thirteen IBVs isolated from chickens in 2005-
2006
(Table 3)
were
assayed using
mRT-PCR. The
genotyping
results
obtained from mRT-PCR were
compared
with those determined
by
the
phylogenetic analysis
of the
partial
S 1
gene
described above.
RESULTS
Multiplex primers.
The
multiplex primer
sets were
designed
from the 5' end of the SI
gene among
IBVs. The
sequences
of the
primers
are listed in Table 2, and the
expected lengths
of the
amplicons
are indicated.
According
to the BLAST
analysis, primers
TWI-R2 and TWII-R2 were
specific
for the TW-I and TW-II
strains,
respectively.
The Conn strain
potentially
annealed with the
primer
H120-R2 at the same site as that of a
Mass-type
strain
(data
not
shown).
IBV
genotyping.
As shown in
Fig.
1, the examined IBVs, TW-I,
TW-II and
Mass-type
strains, were
accurately genotyped by
identifying
the
corresponding
bands on the
agarose gels
(TW-I:
322
bp,
TW-II: 161
bp,
Mass
type:
256
bp)
after mRT-PCR,
which was in
agreement
with the
previous
viral
genome sequence
data.
Mass-type
vaccines
(HI 20, Ma5, and
M41)
and the Conn
strain were identifiable
using
mRT-PCR, whereas the 4/91 vaccine
was not
(Fig. 2).
Detection limit and
accuracy.
The detection limit was
determined
using
the known
copy
number of in vitro transcribed
SI RNA. As indicated in
Fig.
3, the
multiplex genotyping assay
can
detect as low as 103
(1
fg RNA),
105
(100
fg RNA),
and 103
(1
fg
RNA)
viral RNA
copies
of the TW-I, TW-II and
Mass-type
strains,
respectively.
When
templates
were
prepared by mixing
two
types
of
RNA
transcripts,
the detection limits of the TW-I and Mass strains
were not affected in the
presence
of
any
other two
types
at either
ratio. However, the detection limit of the TW-II strain was
decreased 100-fold when the
template
was mixed with 10-fold the
number of RNA
copies
of
any
other two
types.
No bands were
Fig.
1. The
genotyping
mRT-PCR
assay
of IBV strains. Lane M:
100
bp
DNA ladder; lanes 1-3: TW-I strain
(322 bp),
TW-II strain
(161 bp),
H 120
(256 bp);
lane 4:
positive
control; lane 5:
negative
control.
generated
when the
genomic
RNAs of AIV and NDV were tested in
this mRT-PCR
assay (data
not
shown).
Clinical
application.
Based on the
phylogenetic analysis
of the SI
gene
(data
not
shown),
11 of the 13 examined isolates
belonged
to
the TW-I
group, sharing
93.4%- 100% nucleotide
identity. Using
mRT-PCR, those 11 field isolates
(11/11, 100%)
were
correctly
genotyped
as TW-I strains
(Fig. 4).
The other two
untyped
isolates,
3374/05 and 3381/06, were confirmed as the China- and
JMK-like
viral variants,
respectively.
Consistent results were obtained from the
two
genotyping
methods.
DISCUSSION
This work
developed
a
rapid
and sensitive method for
typing
IBV
strains and
discriminating
vaccine strains from
circulating
isolates.
Fig.
2. The
genotyping
mRT-PCR
assay
of IBV vaccines. Lane M:
100
bp
DNA
ladder; lanes 1-3:
Mass-type
vaccines H120, Ma5, M41;
lane 4: Conn strain; lane 5: 4/91 strain; lane 6:
positive
control.
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Multiplex
RT-PCR for IBV
genotyping
107
Fig.
3. The detection limits of the mRT-PCR
assay
were 103
(1 fg
RNA),
105
(100
fg
RNA)
and 103
(1
fg
RNA)
viral RNA
copies
for the
TW-I
(a),
TW-II
(b)
and H 120
(c)
strains. Standard RNA was in vitro-
transcribed and ten-fold
serially
diluted to 102- 10
copies per
5

and
used as
templates
in the mRT-PCR.
Taiwan IBVs as well as most of the current vaccine strains can be
clearly
differentiated
by
their
amplicon
sizes
using
mRT-PCR to
avoid further
sequencing
or
enzyme digestion.
As the
heterologous
vaccines have been used in flocks for a
long period
of time, a DIVA
(differentiating
infected from vaccinated
animals)
diagnostic
test
(30)
is crucial for viral surveillance.
Using
the mRT-PCR
genotyping assay,
viral
samples
can be
quickly diagnosed
as local
infections or vaccine virus interferences. Furthermore, a
variety
of
vaccines,
including Mass-type
strains 120, Ma5, M4l, and Conn
strain, can be identified.
A
uniplex
RT-PCR
assay
was
previously
described to differentiate
between field isolates and vaccine strains
(27).
In that
assay,
different
amplicon
sizes were
expected
based on a 58-base deletion in the
3'UTR of the vaccine strains. However,
using
the
uniplex assay may
be limited when the field isolates fall into more than one
group.
Multiplex
RT-PCR
diagnosis,
which can
target multiple
strains in
one reaction, is more suitable for a
region
with
co-circulating
viruses.
Another
diagnostic multiplex
RT-PCR that
targets
both the SI and
S2
genes
has also been
reported
(26). However, it is unable to
type
the viruses because it uses universal
primers.
In an
attempt
to determine the detection limit of
mRT-PCR,
RNA
transcripts containing
the SI
gene region
of three
targeted
strains were
prepared
to serve as the standard
templates.
The mRT-
PCR
assay
was able to detect as low as 103 viral RNA
copies
of the
TW-I and 120 strains
(equal
to 1
fg RNA).
When
compared
with
other work, the mRT-PCR described here was more sensitive than
another
multiplex
RT-PCR
assay
that detected RNA at a level of
5
pg
(34).
Since different
types
of IBVs are known to exist in the
same
sample
(2),
the detection limit of this
assay
was
additionally
evaluated with
templates
mixed with two
types
of viruses at different
concentrations. The results showed that two of three
types
remained
at the same detection limit. Clinical
samples may
contain
nonspecific
PCR inhibitors
(14)
or a lower viral load, and a more
sensitive
diagnostic assay implies
direct detection from the infected
tissues.
Among
the 13 examined IBV clinical isolates,
11 isolates were
genotyped
as TW-I
group by
the mRT-PCR.
Sequence analyses
confirmed the results of strain
typing.
The other two
untyped
isolates were
subsequently
identified as viral variants
using
full SI
gene sequencing.
One of the viral variants was China-like variant
3374/05 (12),
and the other was a
JMK-like
isolate. IBVs
possess
a
wide
genetic diversity
and are
thought
to mutate at a
high frequency
(24).
In the case of
diagnosis,
mRT-PCR should follow a
generic
IBV detection method, such as an
gene-based
RT-PCR.
Therefore,
false-negative
results from the mRT-PCR can be avoided.
Through
the viral
genotyping diagnosis,
the
untyped
isolates
may
be
reminiscent of the
emergence
of the variants.
The HVRs located in the SI
gene
were
commonly
used in the
identification and characterization of IBVs
(25,28,31,32).
The two
groups
of Taiwan IBVs and
Mass-type
strains showed as little as
79%
identity
in the SI
gene
(17).
In this
study,
the
primer
sets for
the mRT-PCR were selected from the 5' end of the SI
gene
and
bracketed HVRs 1 and 2. In addition to direct differentiation, the
amplicon sequences
from the mRT-PCR
may provide
useful
information to be
analyzed
with the
phylogenetic profile (26,31).
However, the risks associated with
primers designed
close to or in
HVRs should be
carefully regarded,
since the HVRs are
subject
to
change through gene
mutations
(19).
Multiple primer
sets
reacting
in one
single
tube
may magnify
background
noise. To achieve better
accuracy,
the
primer
sets
employed
in mRT-PCR should be
carefully designed.
A hot-start
DNA
polymerase
was
employed
in this
assay
to avoid
any
nonspecific binding
from
multiplex primers. Using
the
specified
equipment
and
reagents,
a whole mRT-PCR reaction can be
completed
within 80 min. Given that DNA
sequencing may
take
several
days,
direct
genotyping
of viral
samples
is
relatively rapid
and
convenient.
The molecular
diagnosis
of IBVs has become universal, and strain
typing
has been a
challenge
in virus research. Here, we
reported
on a
Fig.
4. Clinical
application
of the mRT-PCR
assay.
Eleven field isolates
(11/11, 100%)
were
correctly genotyped
as TW-I strains, and the other
two isolates were
untyped
viral variants. Lanes 1-7, 9, 11-13: TW-I isolates; lane 8: China-like isolate; lane 10:
JMK-like isolate; lane 14:
positive
control.
This content downloaded from 192.188.55.3 on Wed, 15 Oct 2014 16:03:04 PM
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108 H.-W. Chen and C.-H.
Wang
rapid
and reliable mRT-PCR
assay
for the
genotyping
of IBVs. The
mRT-PCR
assay
detected and discriminated vaccine viruses from
wild-type
strains in the field. This
assay may greatly
benefit virus
identification and differentiation in routine disease surveillance.
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ACKNOWLEDGMENTS
We thank the valuable comments
provided
from the two
anonymous
reviewers and Dr. Yuan-Pin
Huang
from the Centers for Disease
Control, Taiwan. The financial
support
from the National Science
Council and the Council of
Agriculture,
Executive Yuan, Taiwan, is
highly appreciated.
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