Development of a Multiplex Quantitative Polymerase Chain Reaction Assay for the Detection of Duck Enteritis Virus, Goose Parvovirus, and Muscovy Duck Parvovirus

Figures

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  Figure 1. Optimization of annealing temperature for multiplex qPCR. During the annealing temperature optimization process, the annealing temperatures were set at 48, 50.2, 51.7, 53.1, 54, 54.6, and 55 °C, respectively. (A) Duck enteritis virus (DEV); (B), Muscovy duck parvovirus (MDPV); (C) Goose parvovirus (GPV).

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  Figure 2. Establishment of singleplex qPCR assay for the individual virus. (A) The amplification curves (left) and a standard curve (right) for detection of DEV UL6 gene were generated concentrations ranging from 1.16 × 108 to 1.16 × 100 copies/μL, y = −3.5197x + 40.21; the coefficient of variation R2 = 0.99; E (amplification efficiency) = 92%; (B) The amplification curves (left) and a standard curve (right) for detection of GPV REP gene were generated concentrations ranging from 9.5 × 108 to 9.5 × 100 copies/μL, y = −3.436x + 37.718; the coefficient of variation R2 = 0.99; E = 95%; (C) The amplification curves (left) and a standard curve (right) for detection of MDPV VP1 gene was generated concentrations ranging from 1.48 × 108 to 1.48 × 100 copies/μL, y = −3.5135x + 39.464; the coefficient of variation R2 = 0.9978; E = 92%.

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  Figure 3. Establishment of multiplex qPCR sensitivity and standard curve. (A) The DEV, MDPV, and GPV amplification curves were generated using multiplex qPCR. Blue indicates the standard curve for DEV; green for MDPV; and red for GPV; (B) Standard curves of multiplex qPCR for detection of DEV, GPV, and MDPV were generated at the optimum amplification conditions. The standard curves of DEV (y = −3.7107x + 38.07 R2 = 0.99 E = 85%), GPV (y = −3.72x + 39.45 R2 = 0.99 E = 85%) and MDPV (y = −3.6923x + 38.346 R2 = 0.99 E = 86%).

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  Figure 4. The specificity of multiplex qPCR. 1–3: plasmid templates of DEV, GPV, and MDPV, 1′–3′: nucleic acid templates of DEV, GPV, and MDPV, 4–9: nucleic acid templates of duck tambusu virus (DTMUV), novel duck reovirus (NDRV), duck hepatitis A type 1 virus (DHAV-1), duck hepatitis A type 3 virus (DHAV-3), avian influenza virus H9 (AIV H9), and Newcastle disease virus (NDV), and nuclease-free water control.