基于RNAscope^(■)原位杂交技术对PRRSV感染组织样本的检测分析

Detection and analysis of porcine reproductive and respiratory syndrome virus in tissue samples based on RNAscope^(■) in situ hybridization

为扩大RNAscope~?原位杂交技术(RNAscope~?ISH)在猪繁殖与呼吸综合征病毒(PRRSV)基础研究中的应用范围,本研究将该技术与免疫组织化学技术(IHC)相结合,通过条件优化建立了RNAscope~?ISH-IHC双重染色技术。经检测不同病毒感染的组织切片中的PRRSV表明该技术具有良好的特异性。采用该双重染色技术检测PRRSV感染的猪淋巴结组织切片中巨噬细胞感染PRRSV情况,结果显示PRRSV核酸主要存在于巨噬细胞胞浆中,在胞核中也有分布,但在一些非巨噬细胞中也散在PRRSV阳性信号,表明PRRSV不仅在猪淋巴结巨噬细胞中复制。利用该双重染色技术检测PRRSV感染后不同时间点猪胸腺组织切片中凋亡相关调节基因Bid、Bcl-xl表达水平,结果显示一段时间内Bid基因转录水平上调、Bcl-xl基因转录水平下调,与同条件下荧光定量PCR结果一致。综上,本研究建立的RNAscope~?ISH-IHC双重染色技术结合了RNAscope~?ISH与IHC各自的优势,不仅能够检测病毒感染后在组织中的定位,还可以分析病毒感染后宿主内源性基因转录的变化趋势,实现了在同一组织切片中对核酸与蛋白的双重检测。经验证该检测方法适用于PRRSV实验室研究,为PRRSV的相关基础研究提供新的检测手段。

To extend the range of RNAscope~? in situ hybridization(RNAscope~?ISH) in porcine reproductive and respiratory syndrome virus(PRRSV) research, our study combined RNAscope~?ISH with immunohistochemistry(IHC) to establish the RNAscope~?ISH-IHC double staining. This method has good specificity in detection of PRRSV in CSFV infected lung section, PRV infected tonsil section and PRRSV infected lymph node section. The PRRSV nucleic acid mainly existed in macrophages cytoplasm and a few in macrophages nucleus, a little in endotheliocyte of PRRSV infected lymph node. These results suggest PRRSV also replicated in other kinds of cells in lymph nodes. We also performed this double staining to detect apoptosis-related genes with the probes targeting Bid or Bcl-xl on thymus sections that infected in different time of PRRSV.Results show the same trend of gene’s regulation to the result real-time fluorescent quantitative PCR detection, indicating there alsowere excellent trend-analysis for the transcription of porcine endogenous gene. In conclusion, our results demonstrate that RNAscope~?ISH-IHC double staining method is reliable, and may be useful in veterinary areas of diagnosis and research.