【摘要】 用Eoehringer Mannheim Biochemica的pAV401探针,以分子杂交的方法鉴定了马铃薯和番茄中的马铃薯纺锤块茎类病毒(PSTV)。试验表明当用探针浓度为200ng/ml, β-半乳糖苷酶为100 mU/ml,即酶结合物稀释至1:100时,可以测定出提纯PSTV—RNA最低浓度为390pg/斑点,感染PSTV的马铃薯和番茄叶片汁液最高稀释浓度为1:128。样品提取方法试验表明,以酚法教果为最佳。健康马铃薯汁液对提纯PSTV—RNA鉴定效果无明显影响。样品汁液在稀释前予先变性对提高杂交效果无明显作用。本文对Boehringer pAV401探针检测PSTV的灵敏度,所用探针浓度以及显色系统进行了讨论和评价。更多还原

【Abstract】 PSTV in infected potato and tomato leaves was detected using pAY 401 DNA probe labeled with a Haptene by RNA/DNA hybridization and enzyme immunoassay according to the Boehringer Mannheim Biochemica’s kit. The minimum amount of PSTV—RNA detected was about 390 pg/per spot. The maximum dilution of infected potato and tomato extract prepared by phenol method, which gives positive reaction, were 1: 128 using. 200mg/ml of pAV 401 DNA—Haptene probe, 100mU/ml of β-galactosidase and 1:100 dilution of antibody-Conjugate. Teats Showed that the routine extraction method with formamide for radioctive detection is not suitable for this nonradioactive detection system. The best results were given by phenol extraction method according to the protocol from the kit. No obvious inhibitory effect of healthy potato sap on detection of PSTV—RNA was observed, and predenaturation of PSTV—infected extracts in 50％ formamide (by heating to 96℃ for 10’ before dilution, and quenching in ice), somehow reduced the positive reaction to some extent. The sensitivity, assay procedure and preparation of sample were discussed.更多还原