Abstract
BackgroundMajor viruses, including duck-origin avian influenza virus, duck-origin Newcastle disease virus, novel duck parvovirus, duck hepatitis A virus, duck Tembusu virus, fowl adenovirus, and duck enteritis virus, pose great harm to ducks and cause enormous economic losses to duck industry. This study aims to establish a multiplex polymerase chain reaction (m-PCR) method for simultaneous detection of these seven viruses.ResultsSpecific primers were designed and synthesized according to the conserved region of seven viral gene sequences. Then, seven recombinant plasmids, as the positive controls, were reconstructed in this study. Within the study, D-optimal design was adopted to optimize PCR parameters. The optimum parameters for m-PCR were annealing temperature at 57 °C, Mg2+ concentration at 4 mM, Taq DNA polymerase concentration at 0.05 U/μL, and dNTP concentration at 0.32 mM. With these optimal parameters, the m-PCR method produced neither cross-reactions among these seven viruses nor nonspecific reactions with other common waterfowl pathogens. The detection limit of m-PCR for each virus was 1 × 104 viral DNA copies/μL. In addition, the m-PCR method could detect a combination of several random viruses in co-infection analysis. Finally, the m-PCR method was successfully applied to clinical samples, and the detection results were consistent with uniplex PCR.ConclusionGiven its rapidity, specificity, sensitivity, and convenience, the established m-PCR method is feasible for simultaneous detection of seven duck-infecting viruses and can be applied to clinical diagnosis of viral infection in ducks.
Highlights
Major viruses, including duck-origin avian influenza virus, duck-origin Newcastle disease virus, novel duck parvovirus, duck hepatitis A virus, duck Tembusu virus, fowl adenovirus, and duck enteritis virus, pose great harm to ducks and cause enormous economic losses to duck industry
We aimed to develop and optimize a single-step multiplex polymerase chain reaction (m-Polymerase Chain Reaction (PCR)) method capable of detecting and differentiating seven major duck viruses, including avian influenza virus (AIV), Newcastle disease virus (NDV), novel duck parvovirus (NDPV), duck hepatitis A virus (DHAV), duck Tembusu virus (DTMUV), fowl adenovirus (FAdV), and duck enteritis virus (DEV)
Optimization and establishment of the m-PCR method A D-optimal design was used to optimize the m-PCR method with 22 runs performed in one randomized batch
Summary
Major viruses, including duck-origin avian influenza virus, duck-origin Newcastle disease virus, novel duck parvovirus, duck hepatitis A virus, duck Tembusu virus, fowl adenovirus, and duck enteritis virus, pose great harm to ducks and cause enormous economic losses to duck industry. Viral infection is one of the most important problems endangering the waterfowls [1]. Highly pathogenic avian influenza could cause high mortality in ducks and geese [3]. Determining AIV in ducks is important in epidemiology research [4]. Similar to AIV, waterfowls are generally considered potential reservoirs for NDV, which has been occasionally reported in China in ducks. Since 1997, Newcastle disease occurred frequently in geese throughout China, causing devastating economic losses [5].
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