The development of a rapid SYBR Green I-based quantitative PCR for detection of Duck circovirus

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.

Evidence of possible vertical transmission of duck circovirus

“…Because of the absence of available cell culture for the isolation of DuCV, the PCR assays were often used for detecting DuCVs (Hattermann et al, 2003;Fringuelli et al, 2005;Jiang et al, 2008;Zhang et al, 2009;Wan et al, 2011;Wang et al, 2011). However, these methods cannot be used to distinguish mixed infection of DuCV-1 and DuCV-2.…”

“…It has been reported that no DuCV specific amplicon was obtained from embryonated duck eggs, non-embryonated duck eggs, and newly hatched ducklings (Wan et al, 2011). In this study, the duplex PCR assay was performed on newly hatched ducklings from eight anti-DuCV antibody positive breeder duck flocks, and duck embryos and non-embryonated eggs from one DuCV-1 and DuCV-2 coinfection flock.…”

Epidemiological investigation of H9 avian influenza virus, Newcastle disease virus, Tembusu virus, goose parvovirus and goose circovirus infection of geese in China

“…Except for GoCV only when virus isolation and the PCR assay were both positive could we confirm that infection was prevalent among the flock. As there are rare reports on avian circovirus being grown in vitro, PCR assays were often used for detecting GoCV (Fringuelli et al., ; Wan et al., ; Wang et al., ; Zhang, Dai, Wang, & Dai, ; Zhang et al., ).…”

A loop-mediated isothermal amplification assay for the visual detection of duck circovirus

“…Other diagnostic techniques, such as conventional polymerase chain reaction (PCR) [2], nested PCR [11], real-time PCR [2,10], ELISA [9] and in situ hybridization (ISH) have been developed. However, these techniques usually need more time and require specialized equipment.…”