Article

Development of a Multiplex Conventional PCR Assay for

Concurrent Detection of FAdV-4, FAdV-8b, and FAdV-11
Su-kyung Kang 1 , Dam-Hee Park 1 , Kyeongcheol Min 1 , Sung-Sik Yoo 1 , In-Joong Yoon 1 and Jongseo Mo 2, *

1 Choong Ang Vaccine Laboratories Co., Ltd., 1476-37 Yuseong-daero Yuseong-gu, Daejeon 34055,
Republic of Korea; [email protected] (S.-k.K.); [email protected] (D.-H.P.);
[email protected] (K.M.); [email protected] (S.-S.Y.); [email protected] (I.-J.Y.)
2 College of Pharmacy, Yeungnam University, Gyeongsan-si 38541, Republic of Korea
* Correspondence: [email protected]; Tel.: +82-053-810-2828

Simple Summary: A multiplex conventional PCR for simultaneously detecting FAdV-4,

-8b, and -11 by targeting the hexon gene was developed. Based on rigorous evaluation, the
assay exhibited high specificity and sensitivity, along with no cross-reactivity among the
target serotypes.

Abstract: Fowl adenovirus (FAdV) belongs to the Aviadenovirus genus within the
Adenoviridae family. FAdVs are widely distributed and associated with various diseases
in poultry, including adenoviral gizzard erosion (AGE), hepatitis-hydropericardium syn-
drome (HHS), and inclusion body hepatitis (IBH). In this study, we developed a multiplex
conventional PCR for simultaneously detecting FAdV-4, -8b, and -11 by targeting the hexon
gene. The multiplex PCR was optimized for primer concentrations and thermocycling
conditions. The optimal primer concentration combination was set at 0.125 µM for FAdV-4,
-8b, and 0.25 µM for FAdV-11. Under these conditions, the limit of detection (LOD) was
103 copies/µL of plasmid standards for FAdV-4, -8b, and -11. These results demonstrated
that the developed multiplex PCR method exhibits high specificity and sensitivity, with no
observed cross-reactivity among these serotypes or with other poultry viruses. Therefore,
Academic Editors: Librado Carrasco this multiplex PCR will be an effective tool for accurate serotyping of FAdV-4, -8b, and -11,
Otero, Ana Margarida Henriques and enabling more precise identification and differentiation of these three serotypes.
Teresa Fagulha

Received: 5 November 2024 Keywords: fowl adenovirus; polymerase chain reaction; multiplex; poultry; diagnosis
Revised: 5 February 2025
Accepted: 10 February 2025
Published: 17 February 2025

Citation: Kang, S.-k.; Park, D.-H.; 1. Introduction

Min, K.; Yoo, S.-S.; Yoon, I.-J.; Mo, J.
Fowl adenovirus (FAdV) is a highly contagious, non-enveloped, double-stranded DNA
Development of a Multiplex
Conventional PCR Assay for
(dsDNA) virus that belongs to the Aviadenovirus genus within the Adenoviridae family. FAdV
Concurrent Detection of FAdV-4, is categorized into five distinct species, each distinguished by unique patterns resulting
FAdV-8b, and FAdV-11. Vet. Sci. 2025, from their restriction fragment length polymorphism (RFLP) profiles [1–3]. FAdV is further
12, 177. https://doi.org/10.3390/ divided into twelve serotypes based on serum neutralization assays (FAdV-1 to 8a, 8b to
vetsci12020177 11) [2]. The whole genome of FAdV has a length of 43–45 kb, encoding several structural
Copyright: © 2025 by the authors. and nonstructural functional proteins [4]. Among the various types of structural genes, the
Licensee MDPI, Basel, Switzerland. hexon gene is notable due to its features as subtype-specific antigenic determinants, making
This article is an open access article it a frequent target for molecular assays for virus detection and routine diagnostics [5].
distributed under the terms and
This is attributed to the hypervariable region in the hexon loop-1 region, allowing the
conditions of the Creative Commons
Attribution (CC BY) license
genetic determination of different FAdV serotypes [6]. In addition, the fiber capsid protein,
(https://creativecommons.org/ known to initiate the cell binding process, is also involved in determining the virulence and
licenses/by/4.0/). tissue tropism of FAdV [7,8]. FAdV is associated with various diseases in poultry, which

Vet. Sci. 2025, 12, 177 https://doi.org/10.3390/vetsci12020177

Vet. Sci. 2025, 12, 177 2 of 13

include adenoviral gizzard erosion (AGE) [9–12], hepatitis-hydropericardium syndrome

(HHS) [13–15], and inclusion body hepatitis (IBH) [16–20]. IBH is characterized by hepatic
necrosis associated with intranuclear inclusion bodies in hepatic cells, while typical lesions
of HHS include accumulation of yellowish clear transudates in the pericardial sac and
necrotic lesions in the liver. Among the various serotypes of FAdV, FAdV-4 from species C
is associated with high mortality rates and evident lesions of HHS. AGE can generally be
observed in chickens infected with FAdV-1 from species A. The age of infection is another
crucial factor, as the severity of the FAdV-associated diseases rapidly increases among
younger chicks [21]. Stand-alone infection [22,23] or co-infection with other immunosup-
pressive viral infections [24–26], such as infectious bursal disease virus (IBDV) and chicken
infectious anemia virus (CIAV), are both possible in most FAdV field cases. Vertical trans-
mission of FAdV from the parental flock to progenies via embryonated eggs is another
critical issue in the industry, making the existence of ‘passed down’ maternal antibodies
important [27–30]. FAdVs are widely distributed worldwide, with different serotypes
prevalent in various countries [31–35], causing significant economic impacts on the poultry
industry. Thus, consistent surveillance and accurate identification of FAdV serotypes are
fundamentally critical. Diagnosis of FAdV infections can be conducted through various
methodologies. Among them, polymerase chain reaction (PCR) assay is still one of the
most conventional yet popular methods in molecular pathology, using a pair of primers
and heat-resistant polymerases to amplify copies of the target DNA [36]. For decades, PCR
has been proven useful for detecting etiologic agents of viral diseases. In some studies, PCR
was used to either simultaneously detect FAdV alongside other avian viruses with FAdV in
a multiplex setting [37,38], or to distinguish FAdV serotypes in a simplex real-time PCR
setting [39]. In this study, to aid in the accurate and reliable diagnosis of FAdV, we present a
conventional multiplex PCR method for the simultaneous detection of FAdV-4 (Species C),
-8b (Species E), and -11 (Species D) that could be conducted on clinical field samples. These
serotypes were selected as they were well known as predisposing, emerging causative
agents for recent FAdV cases worldwide, particularly in Asia [31,40–43]. This assay detects
the presence of the hexon gene for simultaneous serotyping purposes within each sample.
Plasmid DNA standards representing the target sequence of FAdV-4, -8b, and -11 were used
to provide authentic standards for hexon gene detection and quantification. Validation and
statistical evaluation of the developed assays were rigorously carried out.

2. Materials and Methods

2.1. Primer Design for Multiplex PCR
Partial or complete hexon sequences were retrieved from NCBI GenBank (https:
//www.ncbi.nlm.nih.gov/, accessed on 2 May 2023). In this study, forty-four sequences
for FAdV-4, forty-four sequences for FAdV-8b, and thirty-five sequences for FAdV-11 were
used to identify consensus sequences representing each serotype. The retrieved sequences
were aligned using the ClustalW algorithm in MEGA 11 software [44], and consensus
sequences were determined for each FAdV serotype. After generating consensus sequences
for FAdV-4, -8b, and -11, they were aligned, and the mismatched sequences were used for
primer design. The primer designs and sequences are listed in Table 1. The newly designed
primers were further validated by an in-depth in silico evaluation using the BLAST search
tool at NCBI GenBank.
Vet. Sci. 2025, 12, 177 3 of 13

Table 1. Primer sequences for FAdV-4, -8b, and -11.

Target Serotype Sequences (5′ → 3′ ) Product Size

Forward GGC AGA ACG TGT CCG CCT
FAdV-4 209 bp
Reverse ACC GTA AGC GTA ATT GTA CTG A
Forward GGA TCG AGG ACG ATG AAA AC
FAdV-8b 103 bp
Reverse GCG ATT GCC CCA GCT GTT GC
Forward GTC ATC ACG GGT CAG ATG ACA
FAdV-11 426 bp
Reverse CGG CCT GTT TGC CTT CAT GA

2.2. Determining PCR Sensitivity Using Plasmid DNA Standards

Each genomic DNA of FAdV-4 strain ADL190734, FAdV-8b strain ADL190617, and
FAdV-11 strain ADL182047 were used as a template to amplify the target fragments. The
PCR products were then purified and cloned into the pTOP TA V2 vector (Enzynomics,
Daejeon, Republic of Korea) using TOPO-Cloning based on the manufacturer’s protocols.
Each constructed plasmid was transformed into E. coli DH5α and cultured in Terrific Broth
(Sigma-Aldrich, St. Louis, MO, USA) at 37 ◦ C overnight. The plasmids were extracted using
the NucleoBond Xtra Midi Kit (Macherey-Nagel, Düren, Germany), and the concentrations
of each plasmid were measured using NanoDropTM 2000 (Thermofisher, Waltham, MA,
USA). The plasmid copy number was calculated using the following formula: dsDNA
copies = Amount of dsDNA (ng) × Avogadro’s constant (6.022 × 1023 )/Length of dsDNA
(bp) × Conversion factor (1 × 109 ) × Average mass of 1 bp dsDNA (660). To determine
PCR sensitivity, plasmid DNA was serially diluted 10-fold from 1 × 1010 copies/µL to
1 × 100 copies/µL.

2.3. Optimization of Simplex and Multiplex PCR Assay

This assay used 0.25 U/reaction of Taq polymerase (Biofact, Daejeon, Republic of
Korea) and 0.2 mM/reaction of each dNTP in fixed amounts. The master mix BioFACT™
2X Taq PCR Pre-Mix, with Band Helper™ (BioFACT™, Daejeon, Republic of Korea) was
used for the multiplex PCR assay. Thermocycling conditions and primer concentrations
were calibrated using plasmid DNA standards. Plasmid DNAs mimicking each target
serotype were used as templates to optimize the annealing temperature ranging from 56 ◦ C
to 64 ◦ C. A mixture of the plasmids from the three serotypes was used to establish the ideal
primer concentration. PCR was conducted with the following parameters: pre-denaturation
at 98 ◦ C for 5 min, followed by 35 cycles of denaturation at 98 ◦ C for 30 s, annealing at
a range from 56 ◦ C to 64 ◦ C for 30 s, extension at 72 ◦ C for 30 s, and a final extension at
72 ◦ C for 10 min. The PCR products were analyzed by electrophoresis on a 2% agarose
gel stained with RedSafeTM nucleic acid staining solution (Intron, Seongnam, Republic of
Korea). All the plasmid standards and PCR products were validated by Sanger sequencing.

2.4. Specificity of the Simplex and Multiplex PCR Assay

To assess the cross-reactivity of each primer set, simplex PCR targeting the three
serotypes was performed with Leghorn male hepatoma (LMH) cell-cultured supernatants.
Subsequently, to evaluate the specificity of the developed assays, PCR was performed
against other poultry viruses, which included avian metapneumovirus (aMPV), avian in-
fluenza virus (AIV), chicken infectious anemia virus (CIAV), and infectious bronchitis virus
(IBV). Positive PCR results were also added for CIAV, AIV, aMPV, and IBV control viruses.
Vet. Sci. 2025, 12, 177 4 of 13

2.5. Sample Preparation for Multiplex PCR

For the diagnosis of FAdV, liver homogenates, and cloacal swab samples are commonly
used [45–47], while LMH cells and chicken embryo fibroblasts (CEF) are mainly used for
FAdV incubation in in vitro settings [34,47]. Therefore, liver homogenates, cloacal swabs,
and LMH cell-cultured supernatants were used to determine the efficacy of the newly
developed multiplex PCR for clinical applications. The viral strains used in this study
included five isolates of FAdV-4, nine of FAdV-8b, and seven FAdV-11, all of which were
isolated from poultry farms in Korea. These viruses were kindly provided by Avinext
Ltd. (Cheongju, Republic of Korea), a Korean veterinary diagnostic agency. Liver and
cloacal swab samples were collected from SPF chickens challenged with each FAdV isolate
(FAdV-4 strain ADL190734, FAdV-8b strain ADL190617, and FAdV-11 strain ADL182047).
A total of 30 SPF chickens at 9 weeks of age were randomly allocated into three groups
(n = 10), and each group of SPF chickens was challenged with a corresponding FAdV strain
via the intravenous IV route. Challenged birds were monitored for one week, and cloacal
swabs were collected daily. In the event of mortality, necropsies were performed at the time
of death, and tissue samples were collected during the one-week study. Liver tissues were
processed into 10% (w/v) homogenates in PBS, subjected to three cycles of freezing and
thawing, and centrifuged at 8000× g for 10 min. Cloacal swabs were treated in 500 µL of
PBS and centrifuged at 13,000 rpm for 30 s. The supernatants were collected and preserved
at −80 ◦ C. For viral culture, LMH cells were infected with FAdV-4, -8b, and -11 to harvest
virus-containing cell-cultured supernatants. Total DNA was extracted from both cloacal
swab and cell-cultured supernatants using the MagMAXTM Viral RNA Isolation Kit and
the MagMAXTM Express Magnetic Particle Processor (Applied Biosystems, Carlsbad, CA,
USA). The extracted DNA was subsequently stored at −20 ◦ C until use.

2.6. Verification of Primer Specificity by an In-Depth In Silico PCR

Due to the lack of biological specimens from FAdV serotypes within the same species
group as the target serotypes, to further verify the specificity of the multiplex assay
and validate the primer designs, a rigorous in silico PCR analysis was performed us-
ing FastPCR software version 6.9. Hexon gene sequences from FAdV species C (FAdV-4,
-10), species D (FAdV-2, -3, -9, -11), and species E (FAdV-6, -7, -8a, -8b) were extracted
in FASTA format from the National Center for Biotechnology Information’s GenBank
(www.ncbi.nlm.nih.gov; USA, accessed on 15 February 2024) and processed through Fast-
PCR 6.9 software. This evaluation of the multiplex PCR primer design utilized hexon
sequence data from a total of 135 FAdVs (Species C, FAdV-4: 18 strains; FAdV-10: 14 strains;
Species D, FAdV-11: 18 strains, FAdV-2: 13 strains, FAdV-3:13 strains, FAdV-9:10 strains;
Species E, FAdV-8b: 15 strains, FAdV-8a:16 strains, FAdV-6: 7 strains, FAdV-7: 11 strains).

2.7. Ethical Approval

The animal study was approved by the Choong Ang Vaccine Laboratories Institutional
Animal Care and Use Committee (IACUC) (permission number 230728-10).

3. Results
3.1. Primer Design of FAdV-4, FAdV-8b, and FAdV-11
Partial or complete hexon sequences of FAdV-4, -8b, and -11 were retrieved from the
NCBI Genbank (https://www.ncbi.nlm.nih.gov/, accessed on 2 May 2023) The sequence in-
formation is shown in Supplementary Tables S1–S3. The hexon sequences for each serotype
were aligned using the ClustalW algorithm in MEGA 11 software. Subsequently, each
serotype’s consensus sequences were aligned, and mismatched sequences were identified
Vet. Sci. 2025, 12, x FOR PEER REVIEW 5 of 13
Vet. Sci. 2025, 12, 177 5 of 13

each serotype’s consensus sequences were aligned, and mismatched sequences were iden-
(Figure 1). Primer-binding
tified (Figure sites were
1). Primer-binding sitesselected to generate
were selected different
to generate amplicon
different sizes, which
amplicon sizes,
were 209 bp for FAdV-4, 103 bp for FAdV-8b, and 426 bp for FAdV-11, respectively.
which were 209 bp for FAdV-4, 103 bp for FAdV-8b, and 426 bp for FAdV-11, respectively.

Figure 1. Primer design of FAdV-4, -8b and -11. The consensus sequences of FAdV-4, -8b and -11
Figure 1. Primer design of FAdV-4, -8b and -11. The consensus sequences of FAdV-4, -8b and -11
were aligned, and mismatched sequences were identified. The figure shows the primer binding sites
were aligned, and mismatched sequences were identified. The figure shows the primer binding sites
for each FAdV serotype. The PCR amplicon sizes were 212 bp (FAdV-4), 103 bp (FAdV-8b), and 426 bp
for each FAdV serotype. The PCR amplicon sizes were 212 bp (FAdV-4), 103 bp (FAdV-8b), and 426
(FAdV-11), respectively.
bp (FAdV-11), respectively.
3.2. Optimization of Thermocycling Conditions
3.2. Optimization
The optimalofannealing
Thermocycling Conditions
temperature for the simplex PCR was initially determined
usingThe optimal
standard annealing
plasmids fortemperature
each FAdV for the simplex
serotype. PCR was initially
The procedure determined
was conducted us-
across
aing standard plasmids
temperature gradientfor fromeach ◦
56 FAdV
C to 64 ◦
serotype. The procedure
C, increasing was conducted
by increments ◦
of 2 C for across
eacha
PCR reaction.gradient
temperature Each primer
from 56 set°Cwas used
to 64 °C, at a final concentration
increasing by incrementsofof1 2µM. Notably,
°C for the
each PCR
simplex
reaction.PCR
Eachresults
primerwere
set wasconsistent
used at across the entire range
a final concentration of 1ofµM.
annealing
Notably, temperatures
the simplex
from 56 ◦ C to
PCR results 64 ◦consistent
were C, as shown in Figure
across 2A,C.
the entire A combination
range of annealingof standard plasmids
temperatures from 56for
°C
each serotype was used to establish the optimal annealing temperatures
to 64 °C, as shown in Figure 2A,C. A combination of standard plasmids for each serotype for the multiplex
PCR assays,
was used to each at a concentration
establish of 108 copies/µL
the optimal annealing per reaction,
temperatures for the as shown in
multiplex PCR Figure 2D.
assays,
FAdV-11 amplification efficacy increased as the annealing temperature
each at a concentration of 10 copies/µL per reaction, as shown in Figure 2D. FAdV-11
8 gradually rose, with
the final optimal
amplification temperature
efficacy increased being 64 ◦annealing
as the C. In contrast, both FAdV-4
temperature and FAdV-8b
gradually rose, with showed
the fi-
slightly decreased amplification efficiency at 64 ◦ C. At 62 ◦ C, however, all serotypes were
nal optimal temperature being 64 °C. In contrast, both FAdV-4 and FAdV-8b showed
appropriately amplified.
slightly decreased Therefore,
amplification the annealing
efficiency at 64 °C.temperature of 62 ◦ C was
At 62 °C, however, determined
all serotypes to
were
be optimal for the multiplex PCR process.
appropriately amplified. Therefore, the annealing temperature of 62 °C was determined
to be optimal for the multiplex PCR process.
3.3. Assessing the Sensitivity of Multiplex PCR Based on Primer Concentrations
The multiplex PCR assay’s sensitivity was evaluated using a balanced mix of standard
plasmids representing FAdV-4, -8b, and -11. Different primer concentrations were tested
in the multiplex PCR to identify which would yield the highest sensitivity, using four
combinations (Table 2). The sensitivity of multiplex PCR was assessed within the range
of 1010 copies/µL to 10 copies/µL of standard plasmids. The efficacy of the multiplex
PCR varied slightly with different primer concentration combinations. At an equal primer
concentration of 0.5 µM, the limit of detection (LOD) was 105 copies/µL for FAdV-11
and 104 copies/µL for both FAdV-4 and FAdV-8b, respectively (Figure 3A). Moreover, a
decreased primer concentration of 0.125 µM for each set led to reduced sensitivity for
 Vet. Sci. 2025, 12, 177 6 of 13

FAdV-11, with the LOD dropping to 108 copies/µL (Figure 3C). However, when primer
concentrations were standardized to 0.25 µM, or adjusted to 0.125 µM for FAdV-4 and
Vet. Sci. 2025, 12, x FOR PEER REVIEW 6 of 13
FAdV-8b with 0.25 µM for FAdV-11, the multiplex PCR reached its optimal sensitivity,
consistently detecting as few as 104 copies across all three FAdV serotypes (Figure 3B,D).

Figure 2. Optimization of thermocycling conditions. Simplex or multiplex conventional PCR was

Figure 2. Optimization of thermocycling conditions. Simplex or multiplex conventional PCR was
performed using single or mixed plasmid DNA standards of FAdV-4, -8b, and -11, with each primer
performed using single or mixed plasmid DNA standards of FAdV-4, -8b, and -11, with each primer
pair set at a final concentration of 1 µM to determine the annealing temperature. (A–C) Simplex
Vet. Sci. 2025, 12, x FOR PEER REVIEW
pair set for
at aFAdV-4
final concentration 7 of 13
PCR (A), FAdV-8 bof(B),
1 µMandtoFAdV-11
determine
(C).the
(D)annealing
Multiplextemperature. (A–C)the
PCR to determine Simplex
optimal
PCR for FAdV-4
annealing (A), FAdV-8 b (B), and FAdV-11 (C). (D) Multiplex PCR to determine the optimal
temperature.
annealing temperature.

3.3. Assessing the Sensitivity of Multiplex PCR Based on Primer Concentrations

The multiplex PCR assay’s sensitivity was evaluated using a balanced mix of stand-
ard plasmids representing FAdV-4, -8b, and -11. Different primer concentrations were
tested in the multiplex PCR to identify which would yield the highest sensitivity, using
four combinations (Table 2). The sensitivity of multiplex PCR was assessed within the
range of 1010 copies/µL to 10 copies/µL of standard plasmids. The efficacy of the multiplex
PCR varied slightly with different primer concentration combinations. At an equal primer
concentration of 0.5 µM, the limit of detection (LOD) was 105 copies/µL for FAdV-11 and
104 copies/µL for both FAdV-4 and FAdV-8b, respectively (Figure 3A). Moreover, a de-
creased primer concentration of 0.125 µM for each set led to reduced sensitivity for FAdV-
11, with the LOD dropping to 108 copies/µL (Figure 3C). However, when primer concen-
trations were standardized to 0.25 µM, or adjusted to 0.125 µM for FAdV-4 and FAdV-8b
with 0.25 µM for FAdV-11, the multiplex PCR reached its optimal sensitivity, consistently
detecting as few as 104 copies across all three FAdV serotypes (Figure 3B,D).
Figure 3. Assessing the sensitivity of multiplex PCR. Equal amounts of plasmid DNA standards
were 10 0
were mixed
mixed and
and serially
serially diluted,
diluted, spanning
spanning aa10-log
10-logrange
rangefrom
from10 1010to
to10
100 copies/µL.
copies/µL. The
The plasmid
plasmid
Table 2. Combination of multiplex PCR primer concentrations.
mixture
mixture was added to the PCR reaction. The figure shows the amplification results with different
was added to the PCR reaction. The figure shows the amplification results with different
primer concentrations (A–D). The specific
Primer primer concentrations are shown in Table 2; Figure 3A for
Experimental Number primer concentrations (A–D). TheConcentrations per Reaction
specific primer concentrations are shown in Table 2; Figure 3A
no. 1, Figure 3B for no. 2, Figure 3C for no. 3, and Figure 3D for no. 4.
for no. 1,FAdV-4
Figure 3B for no. 2, Figure 3C for FAdV-8b
no. 3, and Figure 3D for no. 4. FAdV-11
1 (Figure 3A) 0.5 µM 0.5 µM 0.5 µM
2 (Figure 3B) 0.25 µM
3.4. Assessing the Specificity of Multiplex0.25
PCRµM 0.25 µM
3 (Figure 3C) 0.125 µM 0.125 µM 0.125
The specificity of the primers was evaluated through both simplex µM
and multiplex
4 (Figure 3D) 0.125 µM 0.125 µM 0.25 µM
PCR. To confirm the cross-reactivity of PCR for each primer set, simplex PCR for FAdV-
4, -8b, and -11 using cell-cultured FAdVs was conducted. The results showed no non-
specific amplification or cross-reactivity with the assigned FAdV primer sets (Figure 4A).
Vet. Sci. 2025, 12, 177 7 of 13

Table 2. Combination of multiplex PCR primer concentrations.

Primer Concentrations per Reaction

Experimental Number
FAdV-4 FAdV-8b FAdV-11
Figure 3. Assessing
1 (Figurethe
3A)sensitivity of multiplex
0.5 µMPCR. Equal amounts
0.5 µMof plasmid DNA 0.5 standards
µM
were mixed and serially diluted, spanning a 10-log range from 1010 to 100 copies/µL. The plasmid
2 (Figure 3B) 0.25 µM 0.25 µM 0.25 µM
mixture was added to the PCR reaction. The figure shows the amplification results with different
3 (Figure 3C) 0.125 µM 0.125 µM 0.125 µM
primer concentrations (A–D). The specific primer concentrations are shown in Table 2; Figure 3A
4 (Figure
for no. 1, Figure 3B for3D)
no. 2, Figure 3C for0.125 and Figure 3D0.125
no. 3,µM µM
for no. 4. 0.25 µM

3.4.
3.4.Assessing
AssessingthetheSpecificity
SpecificityofofMultiplex
MultiplexPCR
PCR
The
The specificity of the primers was evaluated
specificity of the primers was evaluated through
through both
both simplex
simplex andand multiplex
multiplex
PCR.
PCR.To To confirm
confirmthe thecross-reactivity
cross-reactivityofofPCR
PCRfor
foreach
eachprimer
primerset,
set,simplex
simplexPCR PCRfor forFAdV-
FAdV-
4,4, -8b,
-8b, and -11 using cell-cultured FAdVs was conducted. The results showed nonon-
and -11 using cell-cultured FAdVs was conducted. The results showed no non-
specific
specificamplification
amplificationor orcross-reactivity
cross-reactivitywith
withthe
theassigned
assignedFAdV
FAdVprimer
primersetssets(Figure
(Figure4A).
4A).
Additionally,
Additionally, a multiplex PCR analysis was conducted to confirm cross-reactivity for
a multiplex PCR analysis was conducted to confirm cross-reactivity for
other
otherpoultry
poultryviruses;
viruses;aMPV,
aMPV,AIV,
AIV, CIAV,
CIAV, and
and IBV.
IBV. The standard
standard plasmid
plasmid mixture
mixture was
was
used
usedas aspositive
positivecontrols.
controls.TheTheagarose
agarosegel
gelimage
imageshowed
showedthatthatthe
thethree
threetarget
target fragments
fragments
(FAdV-4,
(FAdV-4, -8b, -8b, and
and -11)
-11) were
were successfully
successfullyamplified
amplifiedusing
usingthe
thestandard
standardplasmid
plasmidmixture.
mixture.
Meanwhile,
Meanwhile, other other poultry viruses were not amplified in the multiplex PCR assay
viruses were not amplified in the multiplex PCR assay (Figure (Figure
4B).
4B). Positive
Positive control
control PCR PCR results
results forfor CIAV,
CIAV, AIV,
AIV, aMPV,
aMPV, andand
IBVIBV control
control viruses
viruses areare shown
shown in
in Supplementary
Supplementary Figure S1. Figure S1.

Figure 4. Assessing the specificity of multiplex PCR. The specificity of the primers was evaluated
through
Figure 4. both simplex
Assessing and
the multiplexofPCR.
specificity (A) Simplex
multiplex PCR.PCR
The was conducted
specificity using
of the FAdV-4,
primers was-8b, and -11
evaluated
genomicboth
through DNAsimplex
(gDNA) andmultiplex
and simplex primer set;Simplex
PCR. (A) Lane 1: PCR
Non-template control,using
was conducted LaneFAdV-4,
2: FAdV-4 gDNA,
-8b, and
Lane 3: FAdV-8b gDNA, Lane 4: FAdV-11 gDNA. (B) Genomic DNA from CIAV and complementary
-11 genomic DNA (gDNA) and simplex primer set; Lane 1: Non-template control, Lane 2: FAdV-4
DNA from AIV, aMPV, and IBV were used for the specificity analysis of the PCR; +: FAdV plasmid
gDNA, Lane 3: FAdV-8b gDNA, Lane 4: FAdV-11 gDNA. (B) Genomic DNA from CIAV and com-
DNA standard mixture served as a positive control. NTC: non-template control.
plementary DNA from AIV, aMPV, and IBV were used for the specificity analysis of the PCR; +:
FAdV plasmid DNA
3.5. Evaluating standard mixture
the Multiplex PCR forserved as Purposes
Clinical a positive control. NTC: non-template control.
In this study, we aimed to develop a practical multiplex PCR that can differentiate
FAdV-4, -8b, and -11, for clinical diagnosis. The developed multiplex PCR was evaluated
using liver homogenates, cloacal swabs, LMH cell-cultured supernatants, and other field
FAdV isolates collected from Korean poultry farms. As a result, the multiplex PCR suc-
cessfully detected FAdV-4, -8b, and -11 in various sample types without any non-specific
amplification (Figure 5). Furthermore, to ensure the specificity of our multiplex PCR, it was
tested against various field isolates: four strains of FAdV-4, eight of FAdV-8b, and six of
FAdV-11, respectively. The agarose gel results confirmed successfully amplified target-sized
bands for each serotype without any non-specific band amplification, as shown in Figure 6.
 was tested
Figure 6. against various field isolates: four strains of FAdV-4, eight of FAdV-8b, and six
of FAdV-11, respectively. The agarose gel results confirmed successfully amplified target-
sized bands for each serotype without any non-specific band amplification, as shown in
Vet. Sci. 2025, 12, 177 Figure 6. 8 of 13

Figure 5. Evaluating the practicality of multiplex PCR for clinical purposes. Multiplex PCR was
performed with clinical samples and cell-cultured supernatants. NTC: non-template control; +: the
positive
Figure 5.control. (A) Chicken
Evaluating liver homogenates;
the practicality of multiplexLanePCR 1:forNot-infected, Lane 2:Multiplex
clinical purposes. FAdV-4 PCR infected,
was
Figure
Lane
performed5. Evaluating
3: FAdV-8b
with clinicalthesamples
infected, practicality
Lane 4:and of multiplex
FAdV-11 infected.
cell-cultured PCR for clinical
(B) Cell-cultured
supernatants. NTC:purposes.
supernatantsMultiplex
non-template (sup) PCR
and
control; was
cloa-
+: the
performed
cal
positive with clinical
swab samples;
control. (A)Lane 1:samples
ChickenMock sup,
liver and Lane
cell-cultured
homogenates;2: FAdV-4supernatants.
Lane infected sup,
1: NTC:
Not-infected,Lane non-template
Lane 3: FAdV-8b control;
2: FAdV-4 infected +:
infected, the
sup,
Lane
3:
LaneFAdV-8b
positive infected,
control.
4: FAdV-11 (A)Lane
infected 4:
Chicken FAdV-11
sup, liver infected.
Lanehomogenates;
5: (B) Cell-cultured
Not-infected Lane supernatants
1: Not-infected,
chicken cloacal swab, Lane(sup)
Lanes and cloacal
2: FAdV-4
6–7: swab
infected,
FAdV-4 in-
samples;
Lane 3:
fected Lanecloacal
FAdV-8b
chicken 1: infected,
Mock sup,
swab, LaneLane
Lanes 2: FAdV-4
4: 8–9:
FAdV-11
FAdV-8b infected
infected. (B)
infected sup, Lanecloacal
3: FAdV-8b
Cell-cultured
chicken swab,infected
supernatants
Lanes(sup) sup,
10–11: Lane
and 4:
cloa-
FAdV-
FAdV-11
calinfected
11 infected
swab samples; sup,
chicken Lane Lane 5:
1: Mock
cloacal Not-infected
swab. sup, chicken
Lane 2:
Multiplex cloacal
FAdV-4
PCR swab, Lanes
infected sup,
was executed 6–7: FAdV-4
Lane 3:FAdV-4,
to identify FAdV-8b infected chicken
infected and
FAdV-8b, sup,
cloacal swab, Lanes 8–9: FAdV-8b infected chicken cloacal swab, Lanes 10–11: FAdV-11 infected
Lane 4: FAdV-11
FAdV-11 strains. infected sup, Lane 5: Not-infected chicken cloacal swab, Lanes 6–7: FAdV-4 in-
chicken cloacal swab. Multiplex PCR was executed to identify FAdV-4, FAdV-8b, and FAdV-11 strains.
fected chicken cloacal swab, Lanes 8–9: FAdV-8b infected chicken cloacal swab, Lanes 10–11: FAdV-
11 infected chicken cloacal swab. Multiplex PCR was executed to identify FAdV-4, FAdV-8b, and
FAdV-11 strains.

Figure 6. Assessing the specificity of multiplex PCR. Multiplex PCR confirmed reactivity with other
Figure 6. Assessing
field FAdV theFour
isolates; (A) specificity
strains ofofFAdV-4
multiplex PCR.strains
(B) Eight Multiplex PCR confirmed
of FAdV-8b reactivity
(C) Six strains with
of FAdV-11.
Multiplex
other field PCR
FAdV was executed
isolates; (A) to identify
Four FAdV-4,
strains FAdV-8b,
of FAdV-4 and strains
(B) Eight FAdV-11of strains.
FAdV-8b NTC: non-template
(C) Six strains of
control; +:Multiplex
FAdV-11. the positivePCRcontrol.
was executed to identify FAdV-4, FAdV-8b, and FAdV-11 strains. NTC:
Figure 6. Assessing
non-template control; the specificity
+: the of multiplex PCR. Multiplex PCR confirmed reactivity with
positive control.
3.6. Primer Specificity Verified by an In-Depth In Silico PCR
other field FAdV isolates; (A) Four strains of FAdV-4 (B) Eight strains of FAdV-8b (C) Six strains of
3.6. In theSpecificity
Primer absence ofVerified
biological samples
by an In-DepthforIn
the other
Silico serotypes besides FAdV-4, 8b, and 11,
PCR
FAdV-11. Multiplex PCR was executed to identify FAdV-4, FAdV-8b, and FAdV-11 strains. NTC:
computational PCR analysis was carried out using the FastPCR software 6.9, as reported in
In the absence of biological samples for the other serotypes besides FAdV-4, 8b, and
non-template control; +: the positive control.
Supplementary Table S4. The analysis predicted that the tests would effectively produce am-
11, computational PCR analysis was carried out using the FastPCR software 6.9, as re-
plicons from
3.6. Primer the intended
Specificity FAdV
Verified strains
by S4.
an without
In-Depth anyPCR
In Silico unintended cross-reaction with other
ported in Supplementary Table The analysis predicted that the tests would effectively
serotypes within the same species, indicating the high specificity of the multiplex assays.
produceIn the absence of
amplicons biological
from samplesFAdV
the intended for thestrains
other serotypes
without besides FAdV-4, 8b,
any unintended and
cross-
11, computational PCR analysis was carried out using the FastPCR software 6.9, as re-
4. Discussion
ported in Supplementary Table S4. The analysis predicted that the tests would effectively
Since its first report in 1949, FAdV has had a long history of impacting the worldwide
produce amplicons from the intended FAdV strains without any unintended cross-
poultry industry in the presence of other immunosuppressive diseases or as stand-alone
pathogens for decades, causing severe economic implications [40]. FAdV is distributed
worldwide, including Europe, Asia, and Africa [33–35,47,48]. Considering the primary
routes of infection, FAdV is easily transmitted horizontally through the oral-fecal route [49]
but is also capable of vertical transmission from the parent bird to their offspring [50].
It is crucial to emphasize that this vertical transmission can occur through embryonated
eggs [16], causing high mortality in young chicks. The prevailing understanding of FAdV
infection in the past was that it resulted from secondary infections caused by other immuno-
suppressive infectious diseases, such as IBDV and CIAV. Nonetheless, recent evidence
has emerged to support that FAdV can act as stand-alone pathogens, initiating primary
infections independently. For instance, It has been proven that FAdV-4 can cause HHS
through a single infection [51], while FAdV-8b and -11 have been shown to cause IBH
through primary infections [52]. The role of vaccines is critical in the control of FAdV,
Vet. Sci. 2025, 12, 177 9 of 13

and vaccination is routinely practiced in commercial poultry operations [31]. Therefore,

accurately detecting and differentiating FAdV serotypes within an infected flock is crucial
to devising targeted and effective vaccination strategies. For that purpose, we propose a
multiplex conventional PCR assay that simultaneously detect and differentiate FAdV-4, -8b,
and -11. For stringent verification and standardization, plasmid DNA standards mimicking
each serotype’s target sequences (hexon gene) were utilized. Using such plasmid DNA
provided rigorous criteria for determining LOD and analytical specificity. Additionally, the
assay was further validated with viral DNA extracted from various sample types, including
clinical and research samples and other field isolates. After optimization of the primer
concentrations, the developed multiplex conventional PCR assay performed equivalently
when testing plasmid DNA standards of all three FAdV serotypes, demonstrating linear
detection over a 7-log range (1010 –103 copies/µL) with a LOD of 103 copies/µL for each
serotype (Figure 3B,D). Non-specific amplification among these serotypes or other poultry
viruses was undetected (Figure 4). All of the known positive samples containing the target
serotypes were determined as positive by the multiplex conventional PCR assay, regardless
of the sample type (Figure 5). Nevertheless, it’s essential to recognize that solely detecting
the hexon loop-1 gene may not always be adequate to provide a definitive diagnosis of a
certain serotype. To be more specific, it was observed that the fiber gene, which regulates
the infection route of the FAdV, was also genetically variable among different species of
FAdV, emphasizing the need to develop a diagnostic assay to detect this specific gene [53].
Consequently, a more sophisticated strategy for FAdV identification that examines not
just the hexon gene but also the fiber gene is warranted in the future. It is known that
both genes are subject to antigenic variation under immune pressure, making them highly
variable [54]. There is also increasing evidence to suggest that FAdVs, particularly species
D and E, which include FAdV-11 and 8b targeted in this research, can undergo genetic
recombination as a result of co-infections [53]. Such recombination events between different
serotypes, which often occur in coinfected cells, could complicate diagnostic outcomes.
Coinfections of FAdVs, leading to conditions like IBH or HPS in poultry, have been previ-
ously reported [55,56]. Moreover, recently, in China the presence of twelve serotypes from
all five species were detected from recent outbreaks [57]
Thus, during outbreaks where multiple serotypes may infect simultaneously, such
complexities must be considered when interpreting the results of serotyping tests. However,
as recombination typically occurs within the same species group, it is hoped that misinter-
pretations will be minimal with our currently developed multiplex assay, which targets
FAdVs from distinct species groups (FAdV-4, Species C; FAdV-11, Species D; FAdV-8b,
Species E). Yet, for future development of multiplex PCR assays aimed at serotyping FAdVs,
especially in species groups D and E where recombination is more probable, the inclusion
of new target genes like the aforementioned fiber gene or even from the open-reading
frames (ORF) in the genome should be genuinely considered [53]
Diagnosis of FAdV can be conducted through various detection methodologies such
as gross and histopathological examinations [52], ELISA [50], agarose gel immunodif-
fusion test, indirect immunofluorescence assay and virus neutralization assay [13], and
even transmission electron microscopy [58]. However, these methods are generally time-
consuming, laborious and must carefully address potential cross-reactivity issues between
different serotypes. Several PCR-based assays have been used to detect and differentiate
FAdVs; such as quantitative real-time PCR [39,59–61], PCR-based dot blot [62], high-
resolution melting-curve analysis [63], LAMP (loop-mediated isothermal amplification)
methods [64,65], nanoparticle-assisted PCR assay [66] and digital PCR [67]. Despite their
efficiency, these techniques are limited by the high costs associated with experimental
materials and the demanding requirements for specialized instruments, which are only
Vet. Sci. 2025, 12, 177 10 of 13

sometimes available in diagnostic settings. Besides its high sensitivity, high sequence-
specificity, and functional simplicity, the conventional multiplex PCR assay additionally
offers an economic advantage by enabling viral gene detection using a smaller sample
volume when multiple genetic analyses need to be conducted within the same sample. It
also reduces to use of other PCR reagents. Furthermore, it saves time by eliminating the
need for multiple PCR runs and reduces pipetting errors since the reaction is carried out
within a single tube.
In conclusion, a multiplex conventional PCR assay that can concurrently detect and
differentiate FAdV-4, -8b, and -11 in a single tube reaction was successfully developed,
allowing this assay to be widely used in research and the field. The provision of this
diagnostic assay will facilitate FAdV detection and serotyping in the field

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/vetsci12020177/s1, Figure S1: Positive PCR results for CIAV, AIV,
aMPV, and IBV control viruses; Table S1: Reference of FAdV-4 Hexon gene sequence from NCBI;
Table S2: Reference of FAdV-8b Hexon gene sequence from NCBI; Table S3: Reference of FAdV-11
Hexon gene sequence from NCBI; Table S4: In silico PCR prediction of the developed multiplex assay.

Author Contributions: Conceptualization, S.-k.K.; Methodology; S.-k.K. and D.-H.P.; software, S.-k.K.
and J.M.; validation, S.-k.K. and J.M.; formal analysis, S.-k.K. and J.M.; investigation, S.-k.K.; original
draft preparation, S.-k.K., D.-H.P. and K.M.; writing—review and editing, J.M.; supervision, S.-S.Y.
and I.-J.Y.; project administration, S.-S.Y. and I.-J.Y. All authors have read and agreed to the published
version of the manuscript.

Funding: This work was supported by Choong Ang Vaccine Laboratories.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The raw data supporting the conclusions of this article will be made
available by the authors on request.

Acknowledgments: The authors appreciate Avinext Co., Ltd. for kindly providing the viruses.

Conflicts of Interest: Su-kyung Kang, Dam-Hee Park, Kyeongcheol Min, Sung-Sik Yoon and In-Joong
Yoon come from Choong Ang Vaccine Laboratories Co., Ltd., but have no conflicts of interest.

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