- 期刊
Detection of Porcine Reproductive and Respiratory Syndrome Virus by in situ Hybridization
原位雜交法偵測豬繁殖與呼吸道症候群病毒
摘要
本實驗嘗試使用digoxigenin（DIG）標定的探針探討非放射性原位雜交法是否適用於豬繁殖與呼吸道症候群病毒（PRRSV）之鑑定與診斷。首先，將自台灣本土分散病毒株MD-001中選殖出之核蛋白衣基因構築入質體，再利用T7與T3 RNA聚合酵素轉錄出sense及antisense DIG標定之RNA探針。以此探針和經PRRSV感染的MARC-145細胞株進行雜交。結果顯示，只有當以antisense RNA探針和感染有PRRSV之細胞作用時，可得到強陽性訊號。此探針亦可偵測細胞感染不同來源的病毒株（包括北美株VR2332及歐洲株Lelystad virus）且並無明顯染色強度的差異性。將人工接種PRRSV的無特定病原豬經福馬林固定、石蠟包埋的切片以antisense探針進行原位雜交，結果在接受檢測的組織中發現有廣泛至微量程度的陽性細胞存在。這些陽性細胞包括了淋巴結和肺臟的巨噬細胞及肝臟的Kupffer細胞，該等細胞在過去的報告中均已被證實會被PRRSV感染。本實驗結果顯示，此DIG標定的RNA探針可用於PRRSV感染的例行鑑定。
並列摘要
A non-radioactive in situ hybridization technique based on the digoxigenin (DIG) labeling method was used for the detection of porcine reproductive and respiratory syndrome virus (PRRSV). Both sense and antisense DIG-labeled RNA probes targeted on the nucleocapsid gene of PRRSV were transcribed from a recombinant plasmid constructed from a local isolate (strain MD-001). A strong positive hybridization signal was obtained from a cell line, MARC-145, infected with PRRSV when the antisense RNA probe was used. In addition, the probe can detect cells infected with different strain of virus including US isolate (VR2332) and Lelystad virus without significant difference in signal intensity. Formalin-fixed paraffin-embedded tissues from the specific pathogen free pigs experimentally infected with PRRSV were examined using the same probe. Extensive to minimal positive signal was obtained from various organs examined. The cells showing positive hybridization signals were macrophages (in lymph nodes etc.) and Kupffer cells ( in liver) which were previously reported to be PRRSV permissive cells These results indicate that DIG-labeled RNA probe can be used routinely for the diagnosis of PRRSV infection.