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Journal of Virological Methods 248 (2017) 172–176

Contents lists available at ScienceDirect

Journal of Virological Methods


journal homepage: www.elsevier.com/locate/jviromet

A multiplex PCR for detection of six viruses in ducks T



Yongjuan Wang, Shanyuan Zhu, Weiming Hong, Anping Wang, Weiyong Zuo
Jiangsu Agri-Animal Husbandry Vocational College, Jiang Su Provincial Key Laboratory of Veterinary Bio-Pharmaceutical High Tech Research, Jiangsu, 225300, China

A R T I C L E I N F O A B S T R A C T

Keywords: In this study, six pairs of specific primers that can amplify DNA fragments of different sizes were designed and
Duck synthesized according to viral protein gene sequences published in GenBank. Then, a multiplex PCR method was
Virus established for rapid detection of duck hepatitis virus 1, duck plague virus, duck Tembusu virus, muscovy duck
Multiplex PCR parvovirus, muscovy duck reovirus, and duck H9N2 avian influenza virus, and achieve simple and rapid de-
Detection
tection of viral diseases in ducks. Single PCR was used to confirm primer specificity, and PCR conditions were
optimized to construct a multiplex PCR system. Specificity and sensitivity assays were also developed. The
multiplex PCR was used to detect duck embryos infected with mixed viruses and those with clinically suspected
diseases to verify the feasibility of the multiplex PCR. Results show that the primers can specifically amplify
target fragments, without any cross-amplification with other viruses. The multiplex PCR system can amplify six
DNA fragments from the pooled viral genomes and specifically detect nucleic acids of the six duck susceptible
viruses when the template amount is 102 copies/μl. In addition, the system can be used to detect viral nucleic
acids in duck embryos infected with the six common viruses. The detection results for clinical samples are
consistent with those detected by single PCR. Therefore, the established multiplex PCR method can perform
specific, sensitive, and high-throughput detection of six duck-infecting viruses and can be applied to clinical
identification and diagnosis of viral infection in ducks.

1. Introduction detection and ELISA are time consuming and work exhausting. Im-
muno-electron microscopy requires expensive equipment and high level
Viral infection is one of the important diseases endangering ducks. of virus purification, thereby limiting their potential application in
The recent expansion of the duck industry, the growth of mixed culture clinical diagnosis. PCR can be used for virus detection without virus
models, the enhanced mobility of humans and animals, the poor water isolation. The application of real-time PCR, LAMP and regular PCR in
quality caused by water pollution, and several other factors have cre- the detection of DHV, ARV, MPDV, DPV and the H7 subtype influenza
ated favorable conditions for spreading the virus. The incidence of local virus has been reported (Li et al., 2016; Xu et al., 2012; Wan et al.,
epidemics of viral diseases has significantly increased, and new epi- 2016; Ji and Wang, 2016; Cheng et al., 2007; Hu et al., 2004; Hu et al.,
demics continue to occur, thereby causing serious economic losses to 2015; Xu, 2012), and shows the advantages of high sensitivity, high
the duck industry. To date, the major duck-infecting viruses include speed and safety. However, these approaches cannot be used to identify
duck hepatitis virus 1 (DHAV-1), duck plague virus (DPV), duck or diagnose six viruses at one test. Multiplex PCR (m-PCR) refers to the
Tembusu virus (DTMUV), muscovy duck parvovirus (MPDV), muscovy PCR reactions where two or more primer pairs are added to the same
duck reovirus (ARV), and duck H9N2 low pathogenic avian influenza PCR reaction system and multiple nucleic acid fragments are amplified.
virus (H9N2) (Gao, 2016). An important concern in the duck industry is This method follows the same process and principles as regular PCR,
the construction of a rapid detection method for these viral infections so but with incomparable advantages over regular PCR (Chamberlain
that preventive measures can be implemented as early as possible and et al., 1988; Henegariu et al., 1997). In terms of viral disease detection
economic losses can be reduced. and differential diagnosis, m-PCR can overcome the bottleneck of LAMP
Methods for the detection of these viral infections include virus can only detect a single virus in one reaction, which can simultaneously
isolation, virus identification, serological detection, immuno-electron detect and identify various viruses (Zheng et al., 1995; Kirschberg et al.,
microscopy, ELISA, and PCR techniques (Tang et al., 2015; Wu et al., 2004; Zen et al., 2016; Shi et al., 2016; Xu et al., 2012; Zhang et al.,
2001; Li, 2006; Li et al., 2016; Xu et al., 2012; Wan et al., 2016). Virus 2015; Zeng et al., 2014; Ren et al., 2016). It is also an effective method
isolation and identification are time consuming, whereas serological for rapid diagnosis of mixed-virus infection in clinical practice. To date,


Corresponding author at: East Phoenix Road No. 8, Taizhou, 225300, China.
E-mail address: jsmyjwc@126.com (W. Zuo).

http://dx.doi.org/10.1016/j.jviromet.2017.07.004
Received 19 January 2017; Received in revised form 9 July 2017; Accepted 11 July 2017
Available online 17 July 2017
0166-0934/ © 2017 Published by Elsevier B.V.
Y. Wang et al. Journal of Virological Methods 248 (2017) 172–176

the m-PCR of common duck viral diseases has not been locally or in- Table 1
ternationally reported. Through comparing the sequences of the genes primer sequences in this study.
related to different viral proteins and designing specific PCR primers
Viruses Primer sequences (5′-3′) Target Amplified Concentration
without cross amplification in the conserved region, this study aims to genes fragments of primers
develop a rapid m-PCR detection method for duck viral infection. (bp) (μM)

DHAV-1 F: GTTTGGGAGGCAATGGTT vp1 360 10


2. Materials and methods
R: ATTGAGTCCACATGAACAG
DPV F: GCCAGTGGACAGTTTGA ul6 153 10
2.1. Collection of virus strains and clinical samples R: GGCCAGTTCTCCATTTG
DTMUV F: GGCTTGTTTGGAAAAGG e 514 10
DHAV-1 −SH, DPV-F34, DTMUV-JS804, and MPDV-FJM3 were R: TTCCAGAGTACTTCACTG
MPDV F: GGCATTGCGATTCCCAAT vp1 215 10
supplied by the Shanghai Veterinary Research Institute of the Chinese
R: GAGTCTCTGCCAGTCTC
Academy of Agricultural Sciences. ARV-HNF was supplied by the Fujian ARV F: GCACTCTGGATCCAGTAC c 438 10
Academy of Agricultural Sciences. H9N2-C/SH/F/98 was supplied by R: CAATGGAGAAGCGAACCG
the College of Veterinary Medicine of Yangzhou University. The SPF H9N2 F: CAAACTCCACAGAAACTG ha 837 10
R: CTGACATTGTGGAATGGC
duck embryos were bought from the Harbin Veterinary Research
Institute. ARV-HNF with Yolk sac inoculation was amplified using 4- Note: F, forward primer; R, reverse primer.
day-old duck embryos. DHAV-1-SH, DTMUV-JS804, DPV-F34, H9N2-
C/SH/F/98, and MPDV-FJM3 with the allantoic cavity inoculation were
amplified using 9-day-old duck embryos. The clinical samples were
diseased ducks from duck farms in Jiangsu and Zhejiang. electrophoresis. After purification, the products were cloned to pMD19-
T, named as pMD-DHAV-1, pMD-DPV, pMD-DTMUV, pMD-MPDV,
2.2. Reagents and instruments pMD-ARV and pMD-H9N2, which were sent to Invitrogen Trading
(Shanghai) Co., Ltd. for sequencing to verify the accuracy of the am-
The dNTP, RNA enzyme inhibitor, and the Viral RNA/DNA plified product. Meanwhile, the blank control group without DNA was
Extraction Kit were from TaKaRa (Dalian, China). The 2× Taqmix and also prepared.
DNA molecular weight marker were from Beijing ComWin Biotech Co. The DNA/cDNA mixtures of six viral genomes were used as tem-
Ltd. (China). Plasmid DNA Extract kit and the MMLV Reverse tran- plates for PCR amplification. Each 50 μl reaction system contained 23 μl
scription Kit were from BBI (USA). The HCI buffer, injector, suction of the DNA/cDNA mixture (in sufficient amounts), 1 μl of the forward
pipette, finger tube, and paraffin wax were commercially available. The primer, 1 μl of the reverse primer, and 25 μl of the 2× Taqmix enzyme.
PCR thermal cycler and low-temperature refrigerated centrifuge were The reaction procedure and electrophoresis were as described above.
from Eppendorf. The spectrophotometer was from BIO-RAD. The elec-
trophoresis apparatus and automatic gel image-processing system were 2.6. Construction of the m-PCR system
from the Shanghai Tanon Science & Technology Co. Ltd.
The six pairs of PCR primers were mixed, and then the DNA/cDNA
2.3. Preparation of viral genome mixture of the six viral genomes (in sufficient amounts) were used as
the template. The established single PCR system and procedure, as well
According to the manufacturer’s instructions for the Viral RNA/DNA as re-optimized conditions, were used to set up the 25, 50, and 100 μl
Extraction Kit, the genomes RNA/DNA of the six kinds of viruses were reaction systems, with a final primer concentrations of 0.1, 0.2, and
extracted and dissolved with non-nucleic acid enzymes water. 0.5 μM, as well as annealing temperatures of 48, 50, 52, and 54 °C, for
Concentration and purity of each genome was measured by spectro- 15, 20, 25, and 30 cycles. The PCR products were analyzed by 1.5%
photometry. The RNAs of DHAV-1, DTMUV, ARV and H9N2 were re- agarose gel electrophoresis for comparison with the expected fragment
verse-transcribed into cDNA by MMLV. The DNA/cDNA was stored at size.
−80 °C for future use.
2.7. Specificity of m-PCR
2.4. Design and synthesis of primers
With DNA/cDNAs of various viral genomes as template, the estab-
The published protein sequences of DHAV-1, DPV, DTMUV, MPDV, lished m-PCR method was used for PCR amplification. 50 μl reaction
ARV, and H9N2 in Genbank were compared with the protein sequences system contained 5 μl of the single-virus template, 12 μl of the mixture
of the preserved viruses in our laboratory. Primer Premier V5.0 and of six primer pairs, 25 μl of 2× Taqmix, and 8 μl of deionized water.
Oligo V6.22 were used to evaluate and screen out primers in the con- The reaction conditions were as follows: pre-denaturation at 94 °C for
served domain. Specific primers were designed as listed in Table 1. The 4 min, followed denaturation at 94 °C for 30 s, annealing at 52 °C for
primers were synthesized by Invitrogen Trading (Shanghai) Co., Ltd. 45 s, and extension at 72 °C for 1 min, for 25 cycles, with a final ex-
tension at 72 °C for 7 min. The PCR product was analyzed by 1.5%
2.5. Detection of primer specificity agarose gel electrophoresis. Deionized water was used as the blank
control.
Genome DNA/cDNA of the existing DHAV-1, DPV, DTMUV, MPDV,
ARV and H9N2 were used as template, to amplify the corresponding 2.8. Evaluation of m-PCR sensitivity
target fragments. The total volume of each reaction system was 50 μl,
which contained 5 μl of the single-virus template, 1 μl of the forward Recombinant plasmid corresponding to the six viral genes was ex-
primer, 1 μl of the reverse primer, 25 μl of the 2× Taqmix enzyme, and tracted by plasmid DNA extraction kit. Among them, pMD-DHAV-1,
18 μl of water. PCR amplification was performed as follows: initial pMD-DTMUV, pMD-ARV, and pMD-H9N2 were transcribed to ssRNA in
denaturation at 94 °C for 4 min, followed by denaturation at 94 °C for vitro based on literature [Xie et al.]. The concentration of each ssRNA
30 s, annealing at 52 °C for 45 s, extension at 72 °C for 1 min, for 30 or plasmid was calibrated to 106 copies/μl, and was used to measure the
cycles, with a final extension at 72 °C for 10 min. At the end of reaction, sensitivity of m-PCR by 10-fold gradient dilution. Mixtures of the six
a 5 μl sample of the product was used for 1.5% agarose gel templates with a final concentration of 106 copies/μl were also used to

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Y. Wang et al. Journal of Virological Methods 248 (2017) 172–176

measure the sensitivity of m-PCR by 10-fold gradient dilution. The re-


sult was analyzed by 1.5% agarose gel electrophoresis.

2.9. Initial application of m-PCR

2.9.1. Simulation of mixed virus infection


A mixture of the six viruses was inoculated into the allantoic cavity
of five 9 day-old SPF duck embryos (100 EID50 for each strain).
Embryos and allantoic fluids were collected respectively 72 h after in-
oculation. Followed by aseptic cutting and grinding, an appropriate
amount of PBS was added for homogenate preparing. The homogenate
was centrifuged at 5000 rpm for 10 min after freezing and thawing
three times. The supernatant was suctioned and used as the specimen
for mixed infection. The total genome of each specimen was extracted
Fig. 2. Detection of combinations of 6 viruses by single PCR.
as stipulated in Section 2.3. After reverse transcription, the established
The DNA/cDNA mixtures of six viral genomes were used as templates for PCR amplifi-
m-PCR method was applied. The amplified product was analyzed by cation.
1.5% agarose gel electrophoresis. The uninfected duck embryos were M, 100 bp DNA ladder; 1, PCR product amplified by DPV primer; 2, PCR product am-
set as the blank controls. A mixture of the six viruses was set as positive plified by ARV primer; 3, PCR product amplified by MPDV primer; 4, PCR product am-
control. plified by DHAV-1 primer; 5, PCR product amplified by DTMUV primer; 6, PCR product
amplified by H9N2 primer.

2.9.2. Detection of clinical samples


A total of 87 clinical visceral samples were collected from ducks in
Jiangsu and Zhejiang provinces and homogenized in PBS after aseptic
crushing and grinding. The homogenate was then prepared by 2.9.1
method for genome DNA/RNA extracting as described in Section 2.3.
After reverse transcription, the established method was used for PCR
amplification. The amplified product was analyzed by 1.5% agarose gel
electrophoresis. The products of single PCR amplification with positive
results were sent to Invitrogen Trading (Shanghai) Co., Ltd. for se-
quencing. PBS was set for blank control.

3. Results

3.1. Detection of primer specificity

Specific pairs of primers were used to amplify target gene from


DNA/cDNA of DHAV-1-SH, DPV-F34, DTMUV-JS804, MPDV-FJM3,
ARV-HNF or H9N2-C/SH/F/98 by PCR method. The result showed that
the fragments with expected sizes can be amplified, while negative
control was not amplified (Fig. 1). Recombinant plasmid containing Fig. 3. Electrophoretogram of the m-PCR products.
PCR product sequencing results showed that the amplified 6 gene se- 1, m-PCR products; M, 100 bp DNA ladder.
quences were fully consistent with the purpose gene sequences. The
amplification of the DNA/cDNA mixture with a single primer is shown system and procedure. When the concentration of primer was 0.2 μM
in Fig. 2. Each pairs of primers can only amplify an expected specific and the annealing temperature was 52 °C, in a 50 μl reaction system
band without any non-specific amplification. with 25 cycles, the expected specific band of each virus can be clearly
observed based on electrophoretic analysis (Fig. 3). Other conditions
3.2. Construction of m-PCR system were exactly the same as single PCR.

The m-PCR conditions were optimized according to the single PCR

Fig. 1. Detection of single virus by single PCR.


1, Blank control; 2, PCR products of various viruses; M, 100 bp DNA ladder.

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Y. Wang et al. Journal of Virological Methods 248 (2017) 172–176

Fig. 4. Detection of m-PCR specificity. Fig. 6. Electrophoretogram of the m-PCR products for simulated co-infected duck em-
1, Blank control; 2, m-PCR product of DPV; 3, m-PCR product of MPDV; 4, m-PCR product bryos.
of DHAV-1; 5, m-PCR product of DTMUV; 6, m-PCR product of H9N2; 7, m-PCR product M, 100 bp DNA ladder; 1–5, m-PCR product simulated co- infected duck embryos; 6,
of ARV; M, 100 bp DNA ladder. positive control; 7, blank control.

3.3. Detection of m-PCR specificity

According to the established m-PCR method, mixture of six pairs of plasmid DNA, the minimum detection amounts of DHAV-1, DPV,
primers were used to amplify DNA/cDNA of each virus. The results MPDV, and ARV were 102 copies/μl, while TMUV and H9N2 were 10
showed that mixed primers can only amplify expected fragment for the copies/μl (Fig. 5-2).
corresponding virus without any additional bands. There was also no
band found in the blank control group (Fig. 4).
3.5. Simulation of mixed infection

3.4. Evaluation of m-PCR sensitivity The established m-PCR method was used to detect the six viruses
infected SPF duck embryos. As shown in Fig. 6, the six specific bands
The sensitivity of established m-PCR was as follows: When using with the same size as the positive control were detected from each
ssRNA or plasmid DNA as the single template, the minimum detection sample. In addition, no amplification was observed in the blank control
amounts of HAV-1, DPV, DTMUV, MPDV, ARV and H9N2 were 10 group.
copies/μl (Fig. 5-1). When using the combined template of ssRNA and

Fig. 5. -1 Detection of m-PCR sensitivity.


1–7, m-PCR products of the single template in the
concentration of 100 copies/μl to 106 copies/μl; M,
100 bp DNA ladder.
-2 Detection of m-PCR sensitivity with combined
template.
1–8, m-PCR products of the 6 virus combined tem-
plate in the concentration of 10−1 copies/μl to 106
copies/μl; M, 100 bp DNA ladder.

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Y. Wang et al. Journal of Virological Methods 248 (2017) 172–176

3.6. Detection of clinical samples reaction system. The proposed method can be widely used in rapid
diagnosis of duck viral infections in a short time with good specificity,
Single PCR or m-PCR was used to detect clinical samples. The se- good accuracy, and high sensitivity.
quencing results of single PCR showed that the positive results were the
target fragments of the corresponding viruses. In detail, 53 of them Acknowledgments
were singly infected, including 13 for DHAV-1, 9 for DPV, 10 for
DTMUV, 3 for MPDV, 6 for ARV and 12 for H9N2. Meanwhile, 32 of This work was supported by the Education Department of Jiangsu
them were co-infections, including 7 for DHAV–1 + DPV, 5 for Province (grant no. 16KJB230004), the Talent Support Project in
DHAV–1 + DTMUV, 2 for ARV + H9N2, 5 for H9N2 + DHAV-1, 1 for Jiangsu Province (grant no: NY023), and the Phoenix Talent Project of
H9N2 + DTMUV, 6 for H9N2 + DPV, 1 for MPDV + DPV, 1 for DPV Jiangsu Agri-animal Husbandry Vocational College.
+ DHAV–1 + DTMUV, 2 for H9N2 + ARV + DPV and 2 for
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