建立一种快速 SYBR 一步实时 RT-PCR 检测方法用于检测猪繁殖与呼吸综合征病毒。

The development of a rapid SYBR one step real-time RT-PCR for detection of Porcine Reproductive and Respiratory Syndrome Virus.

机构信息

Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiologic Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China.

出版信息

Virol J. 2010 May 10;7:90. doi: 10.1186/1743-422X-7-90.

BACKGROUND

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. 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.

RESULTS

The 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).

CONCLUSION

The real-time qRT-PCR assay described in this report allows the rapid, specific and sensitive laboratory detection of PRRSV in field samples.

背景

现场样本中 PRRSV 的快速检测对于有效控制 PRRS 非常重要，从而可以减少因暴发而导致的潜在严重经济损失。本研究中，建立了一种基于 SYBR 的快速一步实时 RT-PCR 定量逆转录 PCR（qRT-PCR）检测猪繁殖与呼吸综合征病毒（PRRSV）的方法。引物是根据 PRRSV N 基因高度保守区的序列设计的。

结果

通过 PRRSV ch-1a RNA 生成标准曲线，实现了实时 qRT-PCR 检测的灵敏度。检测限为每个反应混合物 9.6 RNA 拷贝。该检测方法具有良好的内、间重复性，共对 65 份临床样本进行了常规 RT-PCR 和 qRT-PCR 平行检测，PRRSV 的检测率从 60.0%提高到了 76.9%。此外，特异性结果表明，该检测方法可以可靠地区分 PRRSV 与其他猪病病毒，如猪瘟病毒（CSFV）、猪水疱病病毒（SVDV）和猪水疱病病毒（VESV）。