Abstract
This report describes a one-step real-time polymerase chain reaction assay based on SYBR Green I for detection of a broad range of duck circovirus (DuCV). Align with all DuCV complete genome sequences and other Genus Circovirus download from the GenBank (such as goose circovirus, pigeon circovirus), the primers targets to the replicate gene of DuCV were designed. The detection assay was linear in the range of 1.31 × 102-1.31 × 107 copies/μL. The reaction efficiency of the assay using the slope (the slope was -3.349) and the Y-intercept was 37.01 from the linear equation was estimated to be 0.99 and the correlation coefficient (R2) was 0.993. A series of experiments were carried out to assess the reproducibility, sensitivity, and specificity of the assay, following by the low intra-assay and inter-assay CVs for CT values obtained with the standard plasmids. The intra-assay CVs were equal or less than 1.89% and the inter-assay CVs were equal or less than 1.26%. There was no cross-reaction occurred with nucleic acids extracted from RA (Riemerella anatipestifer), E. coli (Escherichia coli), Duck Cholera (Pasteurella multocida), Avian influenza virus, avian paramyxovirus, Muscovy duck parvovirus, Duck reovirus, Duck hepatitis A virus as control templates. The nucleic acids extracted from samples of healthy ducks were used as negative controls. The assay was specific and reproducible. The established real time PCR was used to detect 45 DuCV-negative samples, which were tested using conventional PCR under the developed optimal conditions, each 15 for embryonated eggs, non-embryonated budgerigar eggs, newly hatched duck, the mixture of the lung, liver, spleen which were analysis for the presence of DuCV DNA, to conform that whether the DuCV can be transmitted vertically. Meanwhile, no positive result was shown by the real-time PCR method. The SYBR Green I-based quantitative PCR can therefore be practically used as an alternative diagnostic tool and a screening method for ducks infected with duck circovirus.
Highlights
Circovirus are small, non-enveloped, icosahedral particles with the diameter of about 20 nm, having a circular single-stranded DNA with approximately 2kilobase in genome size [1]
Real-time poly-merase chain reaction (PCR) for duck circovirus (DuCV) Ten-fold serial plasmid dilutions were used to construct the standard curve by plotting the logarithm of the plasmid copy number against the measured cycle threshold (Ct) values (Figure 1 and Figure 2)
Specificity of the assay The specificity of the assay was examined with regard to the nucleic acids extracted from RA (Riemerella anatipestifer), E. coli (Escherichia coli), Duck Cholera (Pasteurella multocida), Avian influenza virus, avian paramyxovirus, Muscovy duck parvovirus, Duck reovirus, Duck hepatitis A virus, were run under the optimal conditions of the assay, and no increase in fluorescence being observed
Summary
Circovirus are small, non-enveloped, icosahedral particles with the diameter of about 20 nm, having a circular single-stranded DNA with approximately 2kilobase in genome size [1]. The family Circoviridae comprised with the two genera Gyrovirus and Circovirus. The genus Gyrovirus contains only the chicken infectious anemia virus (CIAV) [2]. Within the genus Circovirus contains several members, including two porcine circovirus types 1 and 2 (PCV1 and PCV2)[3], the psittactine beak and feather disease virus (BFDV)[4], the columbid circorus (CoCV, known as pigeon circovirus (PiCV)) [5,6], the. Duck circovirus (DuCV) had been listed as a tentative member of the Genus circovirus by ICTV. It was reported first in Germany at 2003 [9]. We firstly reported the detection of DuCV in Fujian Province, China [19]
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