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)
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Y. Wang et al. Journal of Virological Methods 248 (2017) 172–176
3. Results
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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.
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
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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
H9N2 + DPV + DHAV-1. The m-PCR results were matched with single References
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