原位分子杂交检测HFRSVRNA方法研究

Study on Methods of HFRSV RNA Detection by in Situ Hybridization

采用非放射性标记物－地高辛碱性磷酸酶标记肾综合征出血热病毒（ＨＦＲＳＶ）ｃＤＮＡ制备分子探针，检测ＨＦＲＳＶ在鼠肺、恙螨体内分布定位，并对原位分子杂交的各环节进行探讨。结果表明：鼠肺的吞噬细胞、支气管粘膜上皮细胞、血管内皮细胞等均见有阳性颗粒，在恙螨卵巢细胞、中肠及支囊上皮细胞也存在阳性颗粒；在前处理中，明胶硫酸铬钾与多聚赖氨酸的铺片效果相似；室温２ｈ，４２℃１ｈ，４％多聚甲醛处理冰冻切片，可有效防止脱片，且保持良好的细胞形态结构；探针ＰＣＲ标记法优于随机引物标记法；预杂交液加入ｓｐｅｒｍＤＮＡ和ｙｅａｓｔｔＲＮＡ，不用硫酸萄聚糖，可有效地减少背景染色；４２℃杂交１６ｈ。

The prober of hemorrhagic fever with renal syndrome virus(HFRSV) cDNA were labled by nonradioaction Dig alkaline phosphatase to detect HFRSV location in mouse lung and chigger mite,and procedure of in situ hybridization(ISH) were studied.The results show that positive signals were detected in macrophagocyte,vascular endothelial cells of mouse lung and ovary cells,midgut cells of chigger mite.The effection was similar between gelatin chromium potassium sulfate and polylysine in pretreatment,sections were prevented from dropping efficiently at room temperature 2h,42℃ 1h and fixation in 4% paraformaldehyde and kept well structure.Effection of prober labered by PCR was better than by random primed.Background would be light while sperm DNA and yeast tRNA were added in prehybridization without dextran sulfate.Optimal results would come up after hybridized 16h in 42℃ and color developed 7h in color solution.