猪繁殖与呼吸综合征病毒反转录环媒恒温检测方法的建立

Rapid detection of porcine reproductive and respiratory syndrome virus by reverse transcription loop-mediated isothermal amplification

本研究利用PrimerExplorer V4软件设计了针对猪繁殖与呼吸综合征病毒(PRRSV)N基因保守区6个特异性部位的4种引物,建立了PRRSV的反转录环媒恒温检测方法。利用该方法所建立的反应体系在恒温水浴锅中作用1 h即可得到其特有的阶梯状条带,而且猪细小病毒、猪圆环病毒、猪瘟病毒、伪狂犬病毒和健康猪睾丸细胞的扩增结果均为阴性。本方法对PRRSV的最低检出量为1×100个～1×101个基因拷贝。反转录环媒恒温检测方法和普通RT-PCR方法检测临床样品的符合率为96.2%。该方法为现地开展猪繁殖与呼吸综合征的快速诊断和综合防治方案的提出提供了有力的诊断工具。

In order to establish a reverse transcription loop-mediated isothermal amplification （RT-LAMP） method for the detection of porcine reproductive and respiratory syndrome virus （PRRSV）, primers specific to 6 regions of N gene were designed using PrimerExplorer V4. Ladder-like products were produced with those PRRSV positive samples by RT-LAMP, while no product was generated with porcine parvovirus, porcine circovirus, classic swine fever virus, pseudorabies virus and swine testis cells. The assay had a detection limit equivalent to 1 to 10 PRRSV copies/reaction. The sensitivity and specificity of the assay were evaluated by comparison with RT-PCR, by 96.2 % consistence. The specificity and simplicity of the assay could make it a useful tool for the detection of PRRSV infection.