Abstract
BackgroundPrompt detection of PRRSV in the field samples is important for effective PRRS control, thereby reducing the potentially serious economic damage which can result from an outbreak. In this study, a rapid SYBR-based, one step real-time RT-PCR quantitative reverse transcription PCR (qRT-PCR) has been developed for the detection of porcine reproductive and respiratory syndrome virus (PRRSV). Primers were designed based on the sequence of highly conservative region of PRRSV N gene.ResultsThe sensitivity of the real-time qRT-PCR assay was achieved through PRRSV ch-1a RNA for the generation of a standard curve. The detection limit of the assay was found to be 9.6 RNA copies per reaction mixture. This assay had excellent intra- and inter-assay reproducibility as in total 65 field samples were screened for the presence of PRRSV by conventional RT-PCR in parallel with qRT-PCR, and the detection rate increased from 60.0% to 76.9%. Moreover, the specificity result indicated that this assay could reliably differentiate PRRSV from the other swine viral diseases, such as classical swine fever virus (CSFV), swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV).ConclusionThe real-time qRT-PCR assay described in this report allows the rapid, specific and sensitive laboratory detection of PRRSV in field samples.
Highlights
Prompt detection of porcine reproductive and respiratory syndrome virus (PRRSV) in the field samples is important for effective PRRS control, thereby reducing the potentially serious economic damage which can result from an outbreak
Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae [1], and it was characterized by respiratory disease in young pigs and severe reproductive failure in sows, including abortion, stillbirths and weak piglets [2]
Optimization of one step SYBR green real-time reverse transcription-PCR (RT-PCR) The annealing temperature and the primer concentration were optimized (Tables 1 and 2) using the RNA extracted from ch-1a as a positive control
Summary
Prompt detection of PRRSV in the field samples is important for effective PRRS control, thereby reducing the potentially serious economic damage which can result from an outbreak. A rapid SYBR-based, one step real-time RT-PCR quantitative reverse transcription PCR (qRT-PCR) has been developed for the detection of porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV has been recognized as one of the most important pathogens of pigs throughout the world [4] This virus was first confirmed in China in 1996, since the virus has spread widely in China [5,6]. Prompt detection of PRRSV in the field samples is important for effective PRRS control, thereby reducing. The aims of this study were to develop a rapid and sensitive method to detect a wide range of field samples of PRRSV in pigs within a short period of time. We used a one step SYBR green real-time RT-PCR
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