Detection of potyviruses by non-radioactive nucreic acid probe.

Potyvirus是植物病毒中最大的一类，在农业中非常重要。本研究的目的是研制一种特异性的非放射性核酸探针，用于检测各种多病毒及其毒株。从福岛县发生严重藤坏死（tsurugre -byo）的豆科植物（Phaseolus vulgaris）中分离到一株NFU病毒，并与从蚕豆中分离到的大豆黄花叶病毒（byv - sb）进行了比较。根据接种试验、血清学试验和免疫印迹检测结果，鉴定NFU为三叶草黄静脉病毒（CYVV-NFU）毒株。从纯化的病毒制剂中提取CYVV-RNA，并按Gubler和Hoffman方法制备互补DNA。将CDNA连接到PUC19质粒的小位点上。用含CDNA插入的质粒转化大肠杆菌，用CYVV抗血清进行免疫筛选。获得3个免疫阳性克隆。用无放射性化学物质光生物素标记的CYVV- rna CDNA探针进行杂交试验，检测CYVV非常容易、灵敏。对两株芜菁花叶病毒（TUMV）进行了研究。萝卜和白菜菌株是通过宿主范围而不是血清学方法来区分的。获得CDNA克隆并用作非放射性探针。

Potyvirus group is one of the biggest group in plant viruses and very important in agriculture. The purpose of this research is to make a specific nonradioactive nucleic acid probe for detecting each polyvirus and its strains. A virus, designated NFU, isolated from bean plants(Phaseolus vulgaris)with severe vine necrosis(Tsurugare-byo)in Fukushima Prefecture was compared with a seed-borne isolate of bean yellow mosaic virus(BYMV-SB)from broad bean. Based on the results of inoculation test, serological test and western blotting, NFU was identified as a strain of clover yellow vein virus(CYVV-NFU). CYVV-RNA was extracted from purified virus preparations and complementary DNA was prepared by the procedure of Gubler and Hoffman. The CDNA was ligated into Smal site of PUC19 plasmid. The E. coli transformed by plasmids containing CDNA inserts were immunoscreened with CYVV antiserum. Three immunopositive clones were obtained. In hybridization test using CDNA probe of CYVV-RNA labeled with photobiotin, a non-radioactive chemical, CYVV was detected very easily and sensitively. Two strains of turnip mosaic virus(TUMV)were also studied. Radish and cabbage strains were distinguished by host range but not by serological method. The CDNA clones were obtained and used as non-radioactive probes.