RNAscope原位杂交技术检测猪繁殖与呼吸综合征病毒RNA方法的建立

Detection of porcine reproductive and respiratory syndrome virus RNA using RNAscope In situ hybridization

为了直观、高效、特异性地检测猪繁殖与呼吸综合征病毒(PRRSV)感染细胞和组织中PRRSV RNA,本研究根据HP-PRRSV Hu N4株ORF7基因序列设计8对28 bp^36 bp双"Z"结构的寡核苷酸探针(PRRSV-Nprobes),建立了一种新的PRRSV RNA原位杂交检测的方法。该方法能特异性的检测PRRSV感染猪肺泡巨噬细胞(PAMs)中PRRSV RNA,而对猪圆环病毒2型(PCV2)、猪瘟病毒(CSFV)、猪伪狂犬病毒(PRV)感染PAMs中的核酸呈现阴性。应用该方法检测PRRSV感染仔猪肺脏、腹股沟淋巴结和胸腺石蜡包埋组织切片中PRRSV RNA,在感染的腹股沟淋巴结中PRRSV RNA呈现集中分布,而PRRSV RNA在肺脏和胸腺中呈弥漫性分布。该方法可用于细胞和组织中核酸的定位及分布规律的研究,特别是用于病毒含量较少的潜伏感染的检测,为PRRSV实验室检测和致病机理研究奠定了良好的试验基础和技术支持。

To visually, efficiently and specifically detect the genomic RNA of porcine reproductive and respiratory syndrome virus （PRRSV） in the cells and tissues infected with highly pathogentic PRRSV （HP-PRRSV） HuN4, we designed 8 pairs of 28bp-36bp probes with the double ＂Z＂ structure based on the HP-PRRSV HuN4 ORF7 gene. RNAscope, a novel assay based on RNA in situ hybridization （ISH） was developed. This method could specifically detect PRRSV RNA in PRRSV-infected porcine alveolar macrophages（PAMs）, but could not detect the nucleic acids of other virus, such as porcine circovirus type 2 （PCV2）, swine fever virus （CSFV） or porcine pseudorabies virus （PRV）. The PRRSV RNA was detected in PRRSV-infected pig lung, inguinal lymph nodes, and thymus paraffin-embedded tissue sections. The genomic RNA of HP-PRRSV in the lung, inguinal lymph nodes and thymus paraffin-embedded tissues could also be detected using RNAscope method. PRRSV RNA was distributed centrally in infected inguinal lymph nodes, whereas PRRSV RNA was diffusely distributed in the lungs and thymus. The assay was suitable for the study of the localization and distribution of viral genomic RNA in PRRSV-infected PAMs and tissues, especially for the detection of latent infection with a small amount of virus, laying a strong foundation and technical support for the laboratory diagnosis and pathogenic mechanism of PRRSV.