一种直接实时聚合酶链反应检测方法，用于快速高通量检测中国高致病性北美猪繁殖与呼吸综合征病毒，无需 RNA 纯化。

A direct real-time polymerase chain reaction assay for rapid high-throughput detection of highly pathogenic North American porcine reproductive and respiratory syndrome virus in China without RNA purification.

机构信息

College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100 China ; College of Life Sciences, Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen, 518060 China.

Hubei Key Laboratory of Animal Embryo and Molecular Breeding, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan, 430064 China.

出版信息

J Anim Sci Biotechnol. 2014 Oct 2;5(1):45. doi: 10.1186/2049-1891-5-45. eCollection 2014.

BACKGROUND

Porcine reproductive and respiratory syndrome virus (PRRSV), and particularly its highly pathogenic genotype (HP-PRRSV), have caused massive economic losses to the global swine industry.

RESULTS

To rapidly identify HP-PRRSV, we developed a direct real-time reverse transcription polymerase chain reaction method (dRT-PCR) that could detect the virus from serum specimen without the need of RNA purification. Our dRT-PCR assay can be completed in 1.5 h from when a sample is received to obtaining a result. Additionally, the sensitivity of dRT-PCR matched that of conventional reverse transcription PCR (cRT-PCR) that used purified RNA. The lowest detection limit of HP-PRRSV was 6.3 TCID50 using dRT-PCR. We applied dRT-PCR assay to 144 field samples and the results showed strong consistency with those obtained by cRT-PCR. Moreover, the dRT-PCR method was able to tolerate 5-20% (v/v) serum.

CONCLUSIONS

Our dRT-PCR assay allows for easier, faster, more cost-effective and higher throughput detection of HP-PRRSV compared with cRT-PCR methods. To the best of our knowledge, this is the first report to describe a real-time RT-PCR assay capable of detecting PRRSV in crude serum samples without the requirement for purifying RNA. We believe our approach has a great potential for application to other RNA viruses.

背景

猪繁殖与呼吸综合征病毒（PRRSV），特别是其高致病性基因型（HP-PRRSV），给全球养猪业造成了巨大的经济损失。

结果

为了快速鉴定 HP-PRRSV，我们开发了一种直接实时逆转录聚合酶链反应方法（dRT-PCR），该方法无需 RNA 纯化即可从血清标本中检测到病毒。我们的 dRT-PCR 检测法从收到样本到得出结果，整个过程可在 1.5 小时内完成。此外，dRT-PCR 的灵敏度与使用纯化 RNA 的常规逆转录 PCR（cRT-PCR）相当。使用 dRT-PCR 检测 HP-PRRSV 的最低检测限为 6.3 TCID50。我们应用 dRT-PCR 检测法对 144 个现场样本进行了检测，结果与 cRT-PCR 检测结果具有很强的一致性。此外，dRT-PCR 方法能够耐受 5-20%（v/v）的血清。

结论

与 cRT-PCR 方法相比，我们的 dRT-PCR 检测法可更轻松、更快速、更具成本效益且高通量地检测 HP-PRRSV。据我们所知，这是首个描述可在无需 RNA 纯化的情况下，从粗制血清样本中检测 PRRSV 的实时 RT-PCR 检测法的报告。我们相信，我们的方法在应用于其他 RNA 病毒方面具有巨大的潜力。